j.  i 


THE  PARASITIC  AMCEB^E 

OF  MAN 


BY 

CHARLES    F.  CRAIG,  M.D. 

CAPTAIN,    MEDICAL    CORPS,    UNITED   STATES   ARMY 


FROM  THE   BACTERIOLOGICAL     LABORATORY  OP  THE   ARMY   MEDICAL    SCHOOL,   WASHING 

TON,  D.  C.,  AND   THE   ROCKEFELLER   INSTITUTE   FOR   MEDICAL   RESEARCH 

NEW   YORK  CITY 


Published  with  the  Authority  of  the  Surgeon  General 
of  the  United  States  Army 


PHILADELPHIA  &  LONDON 

J.  B.  LIPPINCOTT  COMPANY 

1911 


UNIVERSITY  OF  CALIFORNIA 

LIBRARY 

COLLEGE  OF  AGRICULTURE 
DAVIS 


COPYRIGHT,  1911 
BY  J.  B.  LIPPINCOTT  COMPANY 


UNIVERSITY  FARM 


Printed  by  J.  B.  Lippincott  Company 
The  Washington  Square  Press,  Philadelphia,  U.  S.  A 


PREFACE. 

THE  great  importance  of  amoebic  infections  of 
the  intestine  and  liver  in  man,  and  the  frequency 
with  which  such  infections  occur  in  our  tropical  pos- 
sessions, render  a  work  upon  the  parasitic  amoebae 
of  man  of  interest  to  the  medical  profession  of  the 
United  States,  especially  as  recent  investigations  have 
proven  that  amoebic  dysentery  is  by  no  means  rare, 
even  in  the  temperate  regions  of  our  own  country. 
Much  of  the  literature  treating  of  this  subject  is  in 
German,  French,  and  Italian,  and  but  little  of  it 
has  been  translated  into  English,  while  nearly  all 
of  the  recent  work  done  upon  the  differentiation  of 
species  of  amoebae  has  been  accomplished  by  German 
investigators,  and  published  only  in  German  period- 
icals and  books.  No  monograph  has  appeared  in 
English  giving  in  detail  the  work  accomplished  by 
numerous  students  of  the  amoebae  which  are  parasitic 
in  man,  and  for  this  reason  I  have  thought  that  such 
a  work  would  prove  of  value  to  the  profession  of 
this  country,  especially  to  medical  officers  of  the 
Army,  Navy,  and  Marine  Hospital  Service,  and  to 
public  health  officers,  as  well  as  to  physicians  prac- 
ticing in  the  infected  portions  of  the  United  States. 


W97? 


iv  PREFACE. 

During  my  service  in  the  United  States  military 
hospitals,  notably  the  U.  S.  Army  General  Hospital, 
Presidio  of  San  Francisco,  Cal.,  and  the  U.  S.  Army 
Division  Hospital,  Manila,  P.  I.,  while  a  member  of 
the  U.  S.  Army  Board  for  the  Study  of  Tropical 
Diseases,  material  for  the  study  of  amoebic  dysentery 
was  nearly  always  available,  and  could  be  utilized  to 
the  best  advantage.  Because  of  most  favorable  op- 
portunities I  have  for  nearly  twelve  years  devoted 
much  study  to  the  amoebae  occurring  in  this  form  of 
dysentery  and  much  of  the  data  given  in  this  mono- 
graph is  based  upon  the  personal  observations  of 
hundreds  of  cases  of  amoebic  dysentery,  both  clinically 
and  upon  the  autopsy  table,  and  upon  the  study  of 
thousands  of  preparations  containing  these  organisms. 

Until  the  work  of  Schaudinn,  published  in  1903, 
no  clear  distinction  had  been  made  between  the 
various  species  of  amoebae  infesting  man,  but  he  was 
able  to  differentiate  two  species,  one  causing  a  form 
of  dysentery,  to  which  he  gave  the  name,  Entamceba 
Mstolytica;  and  another,  a  harmless  commensal  of 
man,  which  he  named  Entamceba  coll.  I  was  able  to 
confirm  his  work  while  studying  amoebae  at  the  U.  S. 
Army  General  Hospital  in  San  Francisco,  and  my 
observations  were  published  in  1905,  being  the  first 
published  which  confirmed  Schaudinn' s  work,  so  far 
as  I  know.  Regarding  my  studies  upon  Entamceba 
Mstolytica,  Hartmann,  in  the  Archives  fur  Protis- 


PREFACE.  v 

tenkunde,  Vol.  XVIII,  No.  2,  1909,  p.  207,  says: 
"  Since  the  publication  of  Schaudinn,  Entamoeba 
histolytica  has  been  accurately  described  only  by 
Craig  (1908),  Werner  (1908),  and  myself  (1906- 
1908),  and  Schaudinn's  description  thereby  con- 
firmed." As  a  matter  of  fact,  I  had  confirmed  Schau- 
dinn's description  and  published  my  results  in  1905, 
and  the  paper  referred  to  by  Hartmann  was  only  a 
continuation  of  that  paper.  At  the  same  time  I  was 
able  to  follow  the  life  cycle  of  both  Entamoeba  histo- 
lytica and  Entamoeba  coli  and  to  demonstrate  the 
truth  of  Schaudinn's  description  of  the  methods  of 
reproduction  of  Entamoeba  histolytica.  Regarding 
my  illustrations  of  the  latter  process,  Hartmann 
(ibid.)  says:  "On  the  other  hand,  he  alone,  until 
now,  has  published  illustrations  of  chromidial  and 
cyst  formation  which  are  capable  of  recognition," 
thus  confirming  the  accuracy  of  these  observations. 

More  recently  (1910-1911)  I  have  been  able  to 
confirm  the  observations  of  Viereck  and  Hartmann 
upon  Entamoeba  tetragena  as  regards  its  morphology 
and  life  cycle,  and  have  demonstrated  the  presence 
of  this  species  in  patients  suffering  from  dysentery 
contracted  in  the  Philippine  Islands  and  in  the  United 
States. 

In  the  following  pages  I  have  endeavored  to  give 
a  detailed  description  of  the  various  species  of  amoebae 
which  have  been  described  as  parasitic  in  man, 


vi  PREFACE. 

especially  as  regards  morphology,  life  cycle,  methods 
of  differentiation,  and  relation  to  disease,  and  to  in- 
clude everything  of  value  resulting  from  the  inves- 
tigation of  these  interesting  and  important  parasites. 
I  have  tried  to  be  conservative  in  the  conclusions 
drawn  from  my  own  work  and  that  of  others,  for 
there  are  many  questions  still  unsettled  regarding 
the  status  of  some  of  the  species  of  amoeba*  described, 
and  it  is  more  than  probable  that  further  research 
will  result  in  the  elimination  of  several  species  which 
are  to-day  held  by  some  observers  to  be  valid. 

I  desire  to  here  express  my  deepest  gratitude  to 
Brigadier  General  George  H.  Torney,  Surgeon 
General  of  the  United  States  Army,  for  constant 
encouragement  and  assistance,  and  for  opportunities 
afforded  me  for  research  work,  and  to  Dr.  Simon 
Flexner,  Director  of  the  Rockefeller  Institute  for 
Medical  Research,  for  many  favors  extended  while 
working  in  that  Institute  and  for  his  interest  in  the 
publication  of  this  work.  My  thanks  are  also  due 
Major  Frederick  F.  Russell,  U.  S.  Army,  Director 
of  the  Bacteriological  Laboratories  of  the  Army  Med- 
ical School,  and  to  those  authors  whose  illustrations 
are  here  reproduced,  and  from  whose  investigations  I 
have  profited.  Credit  has  been  given  in  every 
instance,  I  believe,  but  if  not,  pardon  is  asked  for  an 
unintentional  omission. 

CHARLES  F.  CRAIG. 

WASHINGTON,  D.  C.,  July  10,  1911. 


CONTENTS 


CHAPTER  PAGE 

I.  HISTORICAL 1 

II.  GENERAL  MORPHOLOGY  AND  BIOLOGY  OF  AMCEB.E 12 

III.  CLASSIFICATION  AND  NOMENCLATURE 28 

IV.  TECHNIQUE 38 

V.  THE  CULTIVATION  OF  PARASITIC  AMCEB^ 58 

VI.     THE  AMCEBJB  OF  THE  INTESTINAL  TRACT 73-229 

Entamceba  coli:    Distribution.     Morphology.     Reproduc- 
tion   and    Life    Cycle.      Cultivation.      Relation    to 

Disease 73 

Entamceba  histolytica:     Distribution.     Morphology.    Re- 
production and  Life  Cycle.     Cultivation.     Relation 

to  Disease 114 

Entamceba  tetragena:      Distribution.     Morphology.     Re- 
production and  Life  Cycle.    Cultivation.    Relation  to 

Disease 179 

Entamceba  minuta:     Distribution.     Morphology.    Repro- 
duction and  Life  Cycle.    Cultivation.     Relation  to 

Disease 200 

Entamoeba  nipponica:      Distribution.     Morphology.    Re- 
production and  Life  Cycle.     Relation  to  Disease ....   207 
Entamceba  tropicalis:      Morphology  and  Discussion  of  .  .   212 
Entamceba  phagocytoides:    Morphology  and  Discussion  of  213 
Entamoeba  undulans:     Morphology  and  Discussion  of  .  .   214 
Paramoeba  hominis:     Distribution.      Morphology.     Re- 
production and  Life  Cycle.     Relation  to  Disease.  .  .  .   215 

VII.     THE  AMCEB.E  OF  THE  MOUTH 230 

Entamoeba  buccalis:      Distribution.      Morphology.     Re- 
production and  Life  Cycle.     Relation  to  Disease ....   230 
Entamoeba  gingivalis  and  Entamoeba  dentalis:       Discus- 
sion of 232 

VIII.     THE  AMCEB^E  OF  THE  GENITO- URINARY  TRACT 233 

Entamceba  urogenitalis 233 

IX.    THE  AMtEBJE   OCCURRING  IN   EXUDATIONS,    ABSCESSES,  AND 

IN  THE  LUNGS 234 

Entamoeba  miurai:      Discussion  of 234 

Entamoeba  kartulisi:      Discussion  of 234 

Entamceba  pulmonalis:      Discussion  of 235 

BIBLIOGRAPHY 237 

INDEX  OF  AUTHORS 247 

GENERAL   INDEX 249 

vii 


LIST  OF  ILLUSTRATIONS 


FIG.  PAGE 

I.     Diagram    illustrating    the    life    cycle    of    Entamceba    coli. 

(After  Hartmann.)     25 

II.     Entamceba  coli.    (After  Casagrandi  and  Barbagallo.) 86 

III.  Diagram  of  Entamceba  coli.     (Craig.) 94 

IV.  Changes  in  the  form  of  Entamceba  coli  during   amoeboid 

motion.      (Craig.) 94 

V.     Photomicrograph  of  Entamceba  coli  and   Entamceba  histo- 

lytica.     (After  Jin-gens.) 100 

VI.     Multiplication    by    simple    division    in    Entamceba    coli. 

(Craig.) 100 

VII.     Schizogony  of  Entamosba  coli.     (Craig.) 104 

VIII.     Sporogony  of  Entamosba  coli.     (Craig.) 104 

IX.     Diagram  of  Entamosba  histolylica.     (Craig.) 116 

X.     Entamceba  histolytica  (after  Hartmann),  showing  character 

of  the  nucleus  and  karyosome 116 

XI.     Entamosba  histolytica.      (Viereck.) 116 

XII.     Photomicrograph  of  Entamceba  histolytica.    (Gray.) 116 

XIII.  Entamceba  histolytica  from  feces  of  dysentery.     (Jiirgens.)  122 

XIV.  Photomicrograph  of  Entamceba  histolytica.    (After  Jiirgens.)  128 
XV.     Photomicrograph  of  Entamosba  histolytica.    (After  Jiirgens.)   128 

XVI.     Entamosba  histolylica.      (After  Roemer.) 128 

XVII.     Entamceba  histolytica.     (After  Hartmann.) 128 

XVIII.     Reproduction  by  budding  in  Entamosba  histolytica.    (Craig.)  138 
XIX.     Entamosba  histolytica  within  mucous  membrane  of  intestine.  138 
XX.     Section  of  intestine,  showing  numerous  amoebae  within  the 

tissues.     (After  Councilman  and  Lafleur.) 148 

XXI.     Section  of  intestine,  showing  numerous  amoebae  within  the 

tissues.     (After  Councilman  and  Lafleur.) 148 

XXII.     Entamceba  tetragena.     (After  Hartmann.) 184 

XXIII.     Various  stages  in  the  development  of  Entamceba  tetragena. 

(After  Hartmann.)   184 

ix 


x  LIST  OF  ILLUSTRATIONS 

XXIV.     Various  stages  in  the  life  cycle  of  Entamosba   tetragena. 

(After  Viereck.) 188 

XXV.     Vegetative  forms  of  Entamceba  tetragena.     (After  Viereck.)  198 

XXVI.     Encysted  forms  of  Amoeba  Umax 198 

XXVII.     Various  stages  in  the  life  history  of  Entamceba    minuta. 

(After  Elmassian.) 206 

XXVIII.     Diagram  of  the  life  cycle  of  Paramoeba  hominis 220 

XXIX.     Amoebic,  cystic,  and  flagellate  stages  of  Paramoeba  hominis. 

(Craig.) 226 

XXX.     Entamoeba  buccalis.     (After  Hartmann.) 232 


PARASITIC  AMOEBAE 
OF  MAN. 

i. 

HISTORICAL. 

THE  history  of  the  development  of  our  knowledge 
of  the  parasitic  amoebae  of  man  is  of  interest  as 
showing  the  length  of  time  required  in  the  study  of 
this  class  of  parasites  before  very  definite  results 
were  obtained,  and  even  to-day  our  knowledge  is 
far  from  complete  in  regard  to  some  of  the  species 
described  and  their  relation  to  disease. 

Probably  the  first  investigator  to  observe 
amoebae  in  the  feces  of  man  was  Lambl,  of  Prague, 
who  in  1860  described  organisms  occurring  in  the 
feces  of  a  child  which  he  interpreted  as  amoebae,  but 
he  attached  no  great  importance  to  their  presence, 
although  the  child  was  suffering  from  severe  diar- 
rhoea at  the  time.  However,  a  careful  perusal  of 
this  paper  shows  that  he  had  his  suspicions  that 
the  organisms  were  the  cause  of  the  diarrhoea,  so 
that  to  him  belongs  the  credit  of  not  only  first 
describing  the  morphology  of  these  parasites,  but 


2  PARASITIC  AMCEB.E  OF  MAN. 

also  of  demonstrating  their  presence  in  association 
with  diarrhoea.  Following  Lambl  in  1870,  Lewis 
and  Cunningham  found  amcebse  in  the  f eces  of  nearly 
20  per  cent,  of  cholera  patients  studied  in  India. 
These  amoeba?  were  described  by  them  as  large, 
granular,  vacuolated,  amoeboid  organisms,  which  mul- 
tiplied by  gemmation.  They  also  found  them  in 
patients  who  were  suffering  from  other  diseases  and 
in  healthy  individuals.  These  investigators  did  not 
consider  the  parasites  of  any  pathological  significance, 
but  interpreted  them  as  stages  in  the  development 
of  flagellates. 

It  was  not  until  1875  that  a  really  accurate  de- 
scription was  given  of  an  amoeba  occurring  in  man. 
At  that  time  Loesch,  of  St.  Petersburg,  had  the  good 
fortune  to  observe  a  patient  suffering  from  dysentery 
in  whose  feces  numerous  amoeboid  organisms  were 
constantly  found.  The  history  of  this  case  shows 
it  to  have  been  one  of  typical  amoebic  dysentery, 
several  relapses  occurring  at  intervals,  during  which 
the  amoeba?  were  demonstrated  in  the  feces.  The 
description  of  the  organisms,  as  given  by  Loesch, 
makes  it  evident  that  they  were  pathogenic  in  nature 
and  it  is  probable  that  he  actually  studied  Entamceba 
histolytica,  the  pathogenic  species  afterward  differ- 
entiated by  Schaudinn.  To  this  parasite  he  gave 
the  name  "Amoeba  coll."  He  was  successful  in 


HISTORICAL.  3 

reducing  ulceration  of  the  intestine  in  a  dog  which 
he  injected  with  feces  containing  these  organisms,  so 
that  to  this  author  belongs  the  credit  of  first  ex- 
perimentally producing  dysentery  in  the  lower 
animals  by  the  injection  of  material  containing  a 
parasitic  amoeba  of  man.  His  researches  were  fol- 
lowed by  further  work  by  Cunningham  and  by  Koch. 
The  former  author  found  amoebae  in  the  feces  of  both 
healthy  and  diseased  individuals  and  described 
bodies  which  he  considered  to  be  spores,  but  he  did 
not  attach  any  pathological  importance  to  the 
amoebae.  Koch  autopsied  five  cases  of  dysentery  in 
Egypt,  two  of  them  complicated  by  abscess  of  the 
liver.  In  the  ulcers  occurring  in  the  intestine  he 
found  numerous  amoebae  and  in  sections  they  were 
found  at  the  base  of  the  ulcerations.  The  parasites 
were  also  found  in  the  capillaries  of  the  liver,  close 
to  the  abscess  walls.  Koch  considered  that  on 
account  of  their  location  they  bore  some  etiological 
relation  to  the  disease. 

Grassi,  in  1879,  confirmed  the  presence  of  amoebae 
in  human  dejecta,  but  considered  them  harmless 
commensals  as  he  found  them  in  both  health  and 
disease.  His  paper  was  followed  by  those  of  Son- 
sino,  Normand,  Perroncito,  Callandrucio,  and 
Blanchard,  all  confirming  the  presence  of  amoebae 
in  the  stools  of  patients  suffering  from  diarrhoea 
or  dysentery. 


4  PARASITIC  AMGEBJE  OF  MAN. 

In  1886  Kartulis  commenced  the  publication  of 
a  series  of  articles  upon  his  investigations  on  amoebic 
dysentery  as  observed  in  Egypt.  These  contribu- 
tions will  always  rank  as  among  the  most  important 
which  have  been  published  regarding  this  subject. 
In  his  first  paper  he  described  amoebae  which  he 
found  in  all  of  one  hundred  and  fifty  cases  of  amoebic 
dysentery,  and  in  later  publications  he  gave  his  results 
after  the  study  of  five  hundred  cases.  From  his  ex- 
tensive experience  he  concludes  that  the  organisms 
are  the  cause  of  a  form  of  dysentery,  often  asso- 
ciated with  liver  abscess,  and  his  thorough  and  scien- 
tific researches  may  be  said  to  have  finally  established 
the  etiological  relationship  of  amoebse  to  certain  forms 
of  dysentery. 

Kartulis  made  some  attempts  at  cultivation  and 
claimed  to  have  been  successful  in  a  few  instances. 
His  work  was  well  controlled  by  the  examination 
of  sections  prepared  from  the  intestine  of  individuals 
dying  from  tuberculosis,  typhoid  fever,  typhus  fever, 
and  other  diseases.  While  he  found  in  sections  made 
from  the  intestine  of  dysenteric  patients  that  the 
amoebae  were  always  present,  he  never  found  them 
In  sections  from  patients  dying  from  other  diseases. 
Despite  their  great  value  the  researches  of  this  author 
attracted  but  little  attention  in  Europe,  America, 
or  in  the  tropics. 


HISTORICAL.  5 

Shortly  after  the  publication  of  the  first  paper  of 
Kartulis  an  investigation  of  dysentery  at  Prague, 
by  Hlava,  resulted  in  the  finding  of  amoebse  in  sixty 
cases  of  the  disease,  his  description  of  the  organisms 
agreeing  with  that  of  Kartulis.  He  experimented 
upon  dogs  and  cats  by  injecting  feces  containing 
amoebae  into  the  rectum,  obtaining  positive  results 
in  two  dogs  out  of  seventeen  and  in  four  cats  out 
of  six  experimented  upon.  This  paper  was  followed 
by  those  of  Massiutin  and  Pfeiffer,  who  both  found 
amoeba3  in  the  feces  in  cases  of  dysentery. 

In  America  the  first  investigator  to  observe 
amoeba?  in  a  dysentery  case  was  Osier  who,  in  1890, 
in  a  patient  suffering  from  chronic  dysentery  com- 
plicated by  liver  abscess  found  amoebge  in  the  stools 
which  answered  in  their  morphology  to  those  de- 
scribed by  Kartulis.  His  observation  was  followed 
in  the  same  year  by  those  of  Musser  and  Stengel,  and 
in  1891,  by  those  of  Dock,  who  confirmed  the  pres- 
ence of  these  organisms  in  dysenteric  stools. 

In  1891,  Councilman  and  Lafleur  published  their 
classical  monograph  upon  amoebic  dysentery,  in  which 
they  concluded  that  the  disease  is  a  clinical  entity  and 
is  characterized  by  definite  pathological  lesions  due 
to  the  amoebae.  Their  study  was  based  upon  fourteen 
cases  of  amoebic  dysentery,  and  besides  giving  a  most 
excellent  description  of  the  parasite  now  known  as 


6  PARASITIC  AMCEB.E  OF  MAN. 

Entamoeba  histolytica,  they  compared  the  clinical 
differences  between  amoebic  dysentery  and  catarrhal 
dysentery,  as  well  as  the  differences  in  the  patho- 
logical lesions  of  the  two  diseases.  They  proposed 
the  name  Amoeba  dy  sentence  for  the  organism  asso- 
ciated with  the  lesions  which  they  described,  and 
clearly  recognized  that  other,  and  perhaps  non- 
pathogenic  amoeba?,  might  infest  the  intestine  of  man. 
It  is  not  too  much  to  say  that  to  these  authors  we 
owe  a  large  part  of  the  interest  which  has  been  shown 
in  the  study  of  dysentery  during  recent  years. 

From  1891  to  1893  confirmatory  studies  ap- 
peared by  Cahen,  Lutz,  Kovacs,  and  Quincke  and 
Roos.  The  latter  authors,  whose  work  was  done  at 
Kiel,  concluded  that  there  were  at  least  two  varieties 
of  pathogenic  amoeba?  as  well  as  one  variety  which 
was  non-pathogenic.  In  view  of  the  recent  separation 
of  two  species  of  pathogenic  amoeba?,  Entamoeba 
Mstolytica  and  Entamoeba  tetragena,  these  observa- 
tions of  Quincke  and  Roos  are  of  interest,  as  it  is 
possible  that  these  authors  were  the  first  to  differ- 
entiate these  two  species. 

In  1894,  Kruse  and  Pasquale,  who  studied  amoebic 
dysentery  in  Alexandria,  Egypt,  confirmed  the  work 
of  Councilman  and  Lafleur  and  considered  that  there 
existed  two  species  of  amoebae,  one  pathogenic  and 
the  other  a  harmless  inhabitant  of  the  intestinal  tract. 


HISTORICAL.  7 

They  were  able  to  produce  typical  amoebic  dysentery 
in  cats  by  the  injection  into  the  rectum  of  pus  con- 
taining amoebae  obtained  from  a  liver  abscess. 

In  1895,  Celli  and  Fiocca  commenced  the  pub- 
lication of  a  series  of  papers  dealing  with  the  amoebae 
occurring  in  man.  They  did  not  consider  these 
organisms  of  etiological  significance  in  dysentery, 
and  their  results  only  added  to  the  confusion  which 
had  arisen  regarding  the  existence  of  pathogenic  and 
non-pathogenic  species. 

In  1897,  Casagrandi  and  Barbagallo  concluded 
that  none  of  the  amoebae  found  in  the  intestine  of  man 
were  pathogenic,  but  they  separated  several  species 
which  will  be  considered  in  the  chapter  devoted  to 
classification  and  nomenclature. 

The  later  investigations  of  Harris,  in  the  United 
States,  of  Strong  and  Musgrave,  and  of  Musgrave 
and  Clegg  in  the  Philippines,  were  confirmatory  of 
the  etiological  importance  of  amoebae  in  the  produc- 
tion of  dysentery,  especially  that  form  prevalent  in 
the  tropics  and  accompanied  by  abscess  of  the  liver. 
These  investigators  jvere  all  successful  in  producing 
dysentery  in  animals  by  material  containing  the 
amoebae  occurring  in  man.  In  1902,  Jiirgens  pub- 
lished a  monograph  upon  amoebic  dysentery  in  which 
he  differentiated  a  pathogenic  from  a  non-pathogenic 
species,  calling  attention  to  the  difference  in  mor- 


8  PARASITIC  AMCEBJE  OF  MAN. 

phology  of  the  two  parasites  and  the  occurrence  of 
the  pathogenic  species  only  in  cases  of  dysentery. 

Although  up  to  this  time  several  investigators 
had  endeavored  to  separate  the  parasitic  amoebae  of 
man  into  species,  either  pathogenic  or  non-pathogenic, 
it  must  be  admitted  that  until  Schaudinn's  publica- 
tion appeared  in  1903  no  clear  description  had  been 
given  of  specific  differences  in  this  class  of  organisms 
as  it  is  represented  in  man.  The  epoch-making  paper 
of  Schaudinn  gave  the  results  of  his  work  at  Rovigno 
where  he  pursued  a  long  series  of  researches  upon 
these  organisms.  He  clearly  showed  that  there  oc- 
curred in  man  at  least  two  species  of  amoeba  differing 
in  their  morphology,  methods  of  reproduction,  and 
life  cycle;  one,  a  harmless  commensal,  occurring 
both  in  health  and  in  disease,  the  other,  the  cause  of 
a  form  of  dysentery.  He  was  able  to  follow  the 
entire  life  cycle  of  both  these  organisms  and  by  ex- 
periments upon  himself  showed  that  one  was  harmless 
and  that  the  other  when  swallowed  during  a  certain 
stage  of  development  wras  capable  of  producing 
dysentery.  He  suffered  from  two  attacks  of  the 
disease  acquired  in  this  manner  and  four  years  later 
died  as  the  result  of  an  abscess  of  the  sigmoid  flexure, 
in  all  probability  produced  by  the  pathogenic  amoeba 
with  which  he  had  infected  himself.  To  the  harmless 
amoeba  he  gave  the  name  (f  Ent amoeba  coli"  while 


HISTORICAL.  9 

to  the  one  producing  dysentery  he  gave  the  name 
ff Entamceba  Mstolytica"  To  this  investigator  un- 
doubtedly belongs  the  credit  of  the  establishment, 
upon  scientific  grounds,  of  two  species  of  amoeba  in- 
festing the  intestine  of  man. 

In  1905,  while  serving  at  the  U.  S.  Army  General 
Hospital  in  San  Francisco,  California,  where  there 
was  always  abundant  material  for  the  study  of 
amoebic  dysentery,  I  was  able  to  partially  confirm 
Schaudinn's  results  and  later  was  able  to  entirely 
confirm  and  add  to  them. 

For  a  long  time  Schaudinn's  work  was  received 
with  doubt,  but  at  the  present  time  almost  every 
authority,  who  has  had  any  experience  in  the  study 
of  these  organisms,  has  accepted  Schaudinn's  clas- 
sification, and  in  the  recent  work  of  such  zoologists 
as  Liihe,  Braun,  Hartmann,  Minchin,  Stiles,  Wen- 
yon,  Doflein,  and  Calkins,  this  classification  has  re- 
ceived complete  recognition,  while  confirmatory 
papers  have  been  published  by  Hartmann,  Werner, 
Viereck,  Jurgens,  Kartulis,  Simon,  Wenyon,  Fan- 
tham,  and  many  others. 

The  work  of  Schaudinn  had  the  effect  of  stimu- 
lating anew  the  study  of  the  amoebae  parasitic  in  man 
and  since  his  paper  appeared  several  new  species 
have  been  described.  It  is  probable  that  only  a  few 
of  these  are  entitled  to  specific  rank,  but  they  will 


10  PARASITIC  AMOEBAE  OF  MAN. 

all  be  considered  in  this  contribution,  the  data  con- 
cerning each  being  given  so  far  as  it  is  on  record. 
The  most  important  new  species  was  described  by 
Viereck  in  1906,  who  found  an  amoeba,  differing  from 
any  heretofore  described,  in  cases  of  dysentery,  to 
which  he  gave  the  name  Entamoeba  tetragena.  His 
observations  have  been  confirmed  by  Hartmann, 
Prowazek  and  myself,  and  there  can  be  no  doubt  but 
that  this  is  a  distinct  species  of  pathogenic  amoeba.  In 
the  same  year  I  described  another  species  of  amoeba 
possessing  both  a  flagellate  and  an  amoeboid  cycle 
of  development  to  which  the  name  "  Par  amoeba 
hominis"  was  given.  In  1904  Prowazek  described  a 
species  occurring  in  the  mouth  to  which  he  gave  the 
name  Entamoeba  buccalis. 

In  addition  to  the  species  just  mentioned  several 
others  have  been  described,  i.e.,  Entamoeba  tropicalis, 
studied  by  Lesage  in  1908;  Entamoeba  minuta,  de- 
scribed by  Elmassian  in  1909;  and  Entamoeba  nip- 
ponica,  described  by  Koidzumi  in  the  same  year.  In 
addition  to  these  there  are  a  few  so-called  species 
which  possess  little  interest  or  scientific  value  by 
reason  of  imperfect  observation  or  description. 

From  this  summary  of  the  history  of  the  develop- 
ment of  our  knowledge  regarding  the  parasitic 
amoebse  of  man  it  will  be  noted  that  it  has  been  of 
slow  growth,  over  fifty  years  having  elapsed  since 


HISTORICAL. 


11 


the  observation  of  Lambl  of  an  organism  of  this 
nature  in  the  intestinal  discharges  of  a  case  suffering 
from  diarrhoeal  disease.  In  fact,  it  may  be  said  that 
almost  all  of  our  exact  knowledge  concerning  the 
specific  differentiation  of  these  parasites  dates  from 
the  paper  of  Schaudinn  published  in  1908,  and  that 
greater  advance  has  been  made  during  the  past  seven 
years  than  in  the  forty-three  years  preceding.  At 
the  present  time  it  is  definitely  established  that  several 
species  of  amoeba  are  parasitic  in  man,  that  some  of 
them  are  capable  of  producing  typical  pathological 
lesions,  and  that  it  is  possible  to  produce  similar 
lesions  in  some  of  the  lower  animals  by  the  feeding 
or  injection  of  material  containing  these  parasites. 
In  addition  the  entire  life  cycle  of  several  of  the 
species  has  been  worked  out  and  the  differentiation 
of  these  species  is  based  upon  the  life  cycle  as  well 
as  upon  definite  morphological  characteristics. 

In  the  succeeding  pages,  under  the  appropriate 
headings,  further  historical  details  will  be  given  con- 
cerning each  species. 


II. 

GENERAL  MORPHOLOGY  AND  BIOLOGY  OF  AMCEB.E. 

BEFORE  considering  in  detail  the  morphology  and 
life  history  of  the  parasitic  amoebae  of  man  it  is 
necessary  to  review  briefly  the  general  morphology 
and  biology  of  amoebae,  as  ignorance  of  the  structure 
and  life  history  of  saprophytic  organisms  belonging 
to  this  sub-class  of  the  Rhizopoda  has  resulted  in 
great  confusion  in  the  classification  of  the  amoebae 
of  man.  It  should  be  constantly  borne  in  mind  that 
the  species  occurring  in  man  comprise  but  a  minute 
number  when  compared  with  the  multitude  of  species 
which  are  free-living  and  which  are  harmless  and 
incapable  of  a  parasitic  existence. 

The  amoebae  belong  to  the  Protozoa,  subphylum 
Sarcodina,  class  Rhizopoda,  sub-class  Amoebina,  and 
order  Gymnamoebida.  These  organisms  are  unicellu- 
lar in  type,  all  of  their  functions  being  performed 
by  the  single  cell.  They  occupy  the  lowest  position 
in  the  animal  kingdom,  but  in  one  genus,  Paramoeba, 
a  gradation  may  be  traced  to  the  next  higher  sub- 
phylum,  the  Mastigophora.  They  are  found  very 
widely  distributed  as  free-living  forms  and  as  para- 
sites within  man  and  many  of  the  lower  animals. 

12 


MORPHOLOGY  AND  BIOLOGY.  13 

Collections  of  stagnant  water  in  almost  every  region 
abound  in  amoebae,  and  in  the  tropics  almost  every 
source  of  water  supply  is  contaminated  with  these 
organisms.  They  have  also  been  found  upon  green 
vegetables,  especially  salad  vegetables,  and  it  is  not 
at  all  improbable  that  they  occur  on  all  garden 
produce  in  regions  where  human  excrement  is  used 
for  fertilizing  purposes.  Fortunately  only  a  small 
number  of  species  are  pathogenic  for  man,  as  other- 
wise almost  every  inhabitant  of  warm  regions  would 
suffer  from  amoebic  dysentery. 

GENERAL   MORPHOLOGY. 

Roughly  stated  these  organisms  may  be  said  to 
consist  of  a  mass  of  protoplasm  containing  a  nucleus 
and  one  or  more  vacuoles,  which  may  or  may  not 
be  contractile  in  character.  Reproduction  occurs  by 
simple  division,  schizogony,  gemmation,  and  repro- 
duction within  a  cyst.  They  vary  in  size  in  different 
stages  of  development,  measuring  from  5  /*  to  70  t*> 
or  more  in  diameter,  and  are  spherical  or  oval  in 
shape  when  not  in  motion.  The  size  of  the  organism 
alone  cannot  be  used  as  a  method  of  species  differen- 
tiation as  it  varies  greatly  at  different  periods  of 
growth.  All  amoebse  present  for  description  a  cyto- 
plasm and  a  nucleus,  together  with  certain  other 


14  PARASITIC  AMGEB^E  OF  MAN. 

primitive  organs  which  will  be  mentioned  in  the  gen- 
eral description. 

THE  CYTOPLASM. — The  body  of  an  amoeba  is 
composed  of  a  mass  of  cytoplasm  varying  in  appear- 
ance and  structure  in  different  species.  As  a  rule, 
two  distinct  portions  of  this  substance  may  be  recog- 
nized, an  outer  portion,  known  as  the  ectoplasm  and 
which  comprises  the  smaller  portion  of  the  cytoplasm, 
and  an  inner  portion,  known  as  the  endo plasm.  It  is 
in  the  latter  portion  that  the  nucleus  lies  imbedded. 

The  ectoplasm  may  be  well  or  illy  defined  from 
the  endoplasm,  and  in  some  species  is  seen  to  be 
composed  of  minute  granules,  while  in  others  it  ap- 
pears structureless.  In  some,  the  ectoplasm  is  grayish 
in  color  and  veil-like  in  appearance,  while  in  other 
species  it  is  glass-like  in  appearance  and  dense  in 
consistence.  Thus  in  Entamceba  coli  the  ectoplasm 
is  difficult  to  distinguish  from  the  endoplasm  and  is 
of  very  delicate  consistence,  while  in  Entamceba  his- 
tolytica  the  ectoplasm  is  very  distinct,  glass-like  in 
appearance,  and  dense  in  structure.  In  some  species 
the  ectoplasm  can  only  be  differentiated  when  the 
organism  is  in  motion,  while  in  others  it  is  impossible 
to  differentiate  it  at  any  time.  In  a  few  instances  the 
ectoplasm  contains  coarse  granules,  the  nature  of 
which  is  unknown. 

The  Endoplasm. — In  all  species  of  amcebse,  if  a 


MORPHOLOGY  AND  BIOLOGY.  15 

high  power  be  used  in  the  examination,  the  endo- 
plasm  is  observed  to  be  composed  of  a  delicate  mesh- 
work  containing  within  it  numerous  granules  which 
vary  in  size  in  different  species.  These  granules  have 
been  termed  "  microsomes  "  by  some  authorities.  As 
a  matter  of  fact  they  differ  in  nature,  some  being 
due  to  undigested  particles  of  food,  some  to  chromidia, 
some  to  waste  materials,  while  others  consist  of  mi- 
nute crystals  or  oil  droplets.  According  to  Butschl  the 
entire  cytoplasm  of  these  organisms  is  composed  of  a 
fluid  substance  contained  within  a  meshwork  com- 
posed of  another  fluid  of  different  composition. 

The  endoplasm  may  be  more  or  less  refractive 
and  may  contain,  besides  the  nucleus,  one  or  more 
vacuoles ;  threads,  granules,  and  masses  of  chromatin 
(chromidia)  ;  various  crystals  derived  from  the  sur- 
rounding media;  bacteria  of  various  kinds;  and  red 
blood  corpuscles  in  the  case  of  the  pathogenic  amoebae 
of  man. 

The  NUCLEUS  varies  in  size,  structure  and  shape 
in  different  species  of  amoebae,  and  is  of  considerable 
service  in  distinguishing  between  such  species.  A 
visible  nuclear  membrane  is  observed  surrounding  the 
nucleus  in  some  species,  while  in  others  such  a  mem- 
brane cannot  be  distinguished.  In  some  species  a 
distinct  karyosome  can  be  seen,  generally  situated 
near  the  centre  of  the  nucleus,  and  this  sometimes  con- 


16  PARASITIC  AMOEBA  OF  MAN. 

tains  a  minute  centriole  or  centrosome,  and  in  one  or 
two  species  is  of  service  in  differentiation.  Within 
the  nucleus  there  occurs  a  varying  amount  of 
chromatin,  the  arrangement  of  which  is  of  value  in 
distinguishing  species.  In  some  species  the  chromatin 
is  collected  in  masses  upon  the  nuclear  membrane, 
while  in  others  it  is  distributed  throughout  the 
nucleus  in  the  form  of  a  network  enclosing  a  well 
defined  karyosome.  The  amount  of  chromatin  varies 
in  different  species,  in  some  being  almost  absent, 
while  in  others  it  constitutes  the  greater  portion  of  the 
nucleus.  A  certain  amount  of  achromatic  substance 
which  is  unstainable  is  contained  within  the  nucleus. 
In  living  specimens  the  chromatin  appears  as  brightly 
refractile  granules  or  masses  situated  within  the 
nucleus  or  arranged  around  the  nuclear  membrane, 
while  in  stained  specimens  it  takes  the  ruby  red  color 
characteristic  of  this  substance  if  any  modification  of 
the  Romanowsky  stain  is  used. 

Besides  the  nuclear  chromatin  certain  species  of 
amoeba?,  when  stained,  present  a  large  amount  of 
this  substance  lying  within  the  endoplasm.  In  such 
instances  the  chromatin  is  distributed  throughout  the 
endoplasm  in  the  form  of  granules  or  threads  or  as 
irregular  masses  which  may  be  situated  near  the 
periphery  of  the  organism.  The  chromatin  situated 
within  the  endoplasm  is  known  as  chromidia  or  idio- 


MORPHOLOGY  AND  BIOLOGY.  17 

chromidia.  The  amount  of  chromatin  present  within 
the  endoplasm  is  of  considerable  value  in  the  differen- 
tiation of  species. 

Besides  the  nucleus ,  the  endoplasm  may  contain 
one  or  more  vacuoles  which  may  be  contractile  or 
non-contractile  in  character.  The  nature  of  the 
vacuoles  is  of  great  value  in  distinguishing  the  para- 
sitic amoebae  found  in  man  from  the  free-living  forms, 
as  a  contractile  vacuole  is  not  present  in  the  parasitic 
amoebae,  but  is  always  present  in  most  of  the  common 
free-living  species.  Thus  in  the  genus  Entamceba  a 
contractile  vacuole  has  never  been  observed  in  speci- 
mens obtained  from  the  human  intestine  and  ex- 
amined in  the  living  condition,  while  in  Amoeba  Umax, 
and  Amoeba  proteus,  common  free-living  species,  a 
contractile  vacuole  is  always  present.  It  is  significant 
that  the  amoebae  which  have  been  cultivated  from  the 
human  intestine  show  in  the  cultural  forms  a  con- 
tractile vacuole,  so  far  as  I  have  observed.  This  fact 
renders  it  more  than  probable  that  these  amoebae  were 
really  free-living  forms  which  had  been  swallowed 
with  food  or  drink  and  had  passed  through  the  intes- 
tinal canal  in  an  encysted  condition. 

The  number  and  size  of  the  vacuoles  varies  in 
different  species.  Thus  in  Entamceba  coli  there  is 
seldom  more  than  one  or  two  small  vacuoles  present, 
while  in  Entamceba  Mstolytica,  a  number  of  vacuoles 


18  PARASITIC  AMOEBAE  OF  MAN. 

may  be  present,  of  large  size.  In  the  parasitic  species 
found  in  man,  vacuoles  are  generally  present  and  are 
digestive  in  character,  but  they  are  never  observed  to 
pulsate.  Small  masses  of  granular  material  are  often 
seen  within  the  vacuoles  and  sometimes  stain  with 
neutral  red,  thus  showing  that  the  vacuoles  are  diges- 
tive in  character.  As  a  rule  the  vacuoles  are  situated 
near  the  periphery  of  the  amoeba?,  especially  when  they 
are  contractile,  but  they  change  their  position  with  the 
movements  of  the  organism.  In  Amoeba  proteus,  a 
free-living  form,  it  is  easy  to  observe  the  formation 
and  contraction  of  the  vacuole.  At  first  it  is  situated 
near  the  nucleus  and  is  very  small,  but  as  nutritive  ma- 
terial collects  within  it,  the  vacuole  grows  larger,  at 
the  same  time  moving  toward  the  periphery  of  the 
organism.  When  it  arrives  at  the  periphery  it  sud- 
denly contracts  and  its  contents  are  rejected  from 
the  body  of  the  amoeba.  Such  phenomena  are  never 
observed  in  the  parasitic  amoebae  of  man. 

In  addition  to  the  vacuoles  there  are  present  in 
the  endoplasm  of  some  species  of  amoeba*,  especially 
those  parasitic  in  man,  numerous  minute,  oval  or 
round  bodies,  which  resemble  the  spores  of  malarial 
plasmodia,  in  unstained  preparations,  and  which  have 
been  described  by  different  authors  as  spores.  At  a 
certain  stage  of  the  development  of  Entamceba  coli, 
several  such  bodies  may  be  observed  in  both  living  and 


MORPHOLOGY  AND  BIOLOGY.  19 

stained  specimens  and  represent  the  nuclei  of  the 
daughter  amoeba?  which  are  forming  within  the  parent 
organism.  In  Entamceba  lustolytica  these  bodies  are 
often  numerous  and  represent  the  collections  of  idio- 
chromidia  which  form  the  nuclei  of  the  young  para- 
sites which  are  produced  by  gemmation. 

Besides  the  vacuoles  and  the  spore-like  bodies  just 
noted,  there  may  occur,  in  the  endoplasm  of  amoebae, 
minute  crystals  of  various  kinds  which  have  been 
taken  up  from  the  surrounding  media,  as  well  as 
bacteria  and  foreign  particles  of  varying  nature.  In 
the  parasitic  amoebae  found  in  man  the  endoplasm 
often  contains  red  blood  cells  if  intestinal  inflamma- 
tion be  present. 

GENERAL   BIOLOGY. 

Amoebae  obtain  nutriment  by  means  of  pseudo- 
podia  which  are  protruded  from  the  body  and  sur- 
round food  materials  which  are  then  taken  into  the 
body  of  the  organism  where  they  undergo  digestion. 
In  these  organisms  we  meet  with  the  most  primitive 
form  of  digestion,  any  portion  of  the  cytoplasm  being 
capable  of  absorbing  and  digesting  food  materials. 
In  those  amoebae  which  possess  a  contractile  vacuole 
the  food  material  is  enclosed  within  it  and  digested,  the 
residue  being  expelled  when  the  vacuole  contracts.  In 
those  organisms  in  which  a  contractile  vacuole  is  absent 


20  PARASITIC  AMOEBAE  OF  MAN. 

there  occur  numerous  digestive  vacuoles  which  per- 
form the  same  function.  In  many  species  of  amoebae, 
especially  those  belonging  to  the  genus  Entamceba, 
the  exact  method  by  which  undigested  food  is  gotten 
rid  of  is  still  unknown,  but  it  is  probable  that  such 
material  is  simply  extruded  from  the  periphery  of 
the  organism. 

RESPONSE  TO  STIMULATION. — Amoebae  are  capable 
of  responding  to  stimulation  of  either  mechanical, 
chemical,  or  electrical  nature. 

Mechanical  stimulation  is  best  illustrated  by  the 
production  of  pseudopodia  when  the  organism  touches 
surfaces  of  any  kind,  and  the  withdrawing  of  the 
pseudopodia  when  it  is  touched  with  a  needle  or  when 
it  comes  in  contact  with  obstacles  to  locomotion. 

Response  to  chemical  irritation  is  noted  when  a 
solution  containing  some  substance  poisonous  to  the 
organism  is  allowed  to  come  in  contact  with  it.  In 
such  instances  the  pseudopodia  are  quickly  with- 
drawn and  the  amoeba  assumes  a  spherical  shape. 

Response  to  electrical  stimulation  is  noted  when 
galvanic  currents  are  passed  through  water  contain- 
ing amoebae.  The  organisms  which  are  motile  quickly 
cease  motion,  the  pseudopodia  are  withdrawn  and  a 
spherical  form  is  assumed.  If  the  currents  are  of  a 
mild  character  the  organisms  renew  their  movements 
in  a  variable  time,  but  if  too  strong,  death  ensues. 


MORPHOLOGY  AND  BIOLOGY.  21 

If  an  amoeba  in  which  several  pseudopodia  are 
present  is  gently  touched  with  a  needle  upon  only  one 
of  the  pseudopodia,  they  are  all  quickly  withdrawn, 
thus  proving  that  the  protoplasm  of  the  organism 
possesses  conductivity. 

MOTILITY. — Aside  from  reproduction,  the  most 
striking  phenomenon  observed  in  amoeba?  is  the  power 
of  locomotion.  This  is  rendered  possible  by  a  form 
of  motility  which,  because  it  is  best  illustrated  in 
amoebae,  is  known  as  amoeboid  motion.  This,  the  most 
primitive  form  of  locomotion,  consists  in  the  throw- 
ing out  of  pseudopodia  at  the  periphery  of  the 
organism  and  the  flowing  into  them  of  the  cytoplasm. 
Jennings  compares  it  to  rolling,  the  upper  surface 
of  the  cytoplasm  passing  forward  and  turning  under 
at  the  anterior  end  of  the  pseudopodia,  thus  produc- 
ing the  appearance  of  the  cytoplasm  flowing  in  the 
direction  of  motion.  By  this  means  an  amoeba  is  able 
to  move  in  any  direction,  the  rate  of  rapidity  of 
motion  varying  with  the  surroundings  and  the  species 
of  organism.  Locomotion  in  any  one  direction  only 
lasts  for  a  short  period  of  time,  and  it  is  not  unusual 
to  observe  an  amoeba  move  in  the  opposite  direction 
without  any  apparent  reason.  When  an  obstacle  is 
encountered  by  a  pseudopodium  it  is  quickly  with- 
drawn and  another  is  protruded,  the  organism  mov- 
ing away  in  another  direction. 


22  PARASITIC  AMCEB^E  OF  MAN. 

The  protrusion  of  pseudopodia  is  not  always  fol- 
lowed by  locomotion  for  organisms  are  frequently 
observed  from  which  pseudopodia  are  protruded  con- 
stantly and  as  constantly  withdrawn,  but  there  occurs 
no  change  in  position.  The  pathogenic  amoebas  are 
generally  more  actively  motile  than  the  non-patho- 
genic species. 

The  pseudopodia  are  always  formed  by  the  ecto- 
plasm and  are  well  defined  in  some  species,  while  in 
others  no  differentiation  can  be  made  between  the 
pseudopodium  and  the  endoplasm.  This  difference 
in  the  appearance  of  the  pseudopodia  is  of  some  value 
in  distinguishing  species. 

Pseudopodia  vary  greatly  in  size  and  shape  in 
different  species,  in  some  being  spinose  and  very 
delicate  in  appearance ;  in  others  finger-like  and  dense 
in  structure;  while  in  still  others  they  are  broad  and 
blunt.  In  some  amcebse  they  are  so  characteristic 
in  appearance  as  to  be  of  great  service  in  species 
differentiation.  Thus  in  Entamoeba  coli  the  pseudo- 
podia are  blunt  and  of  a  delicate  veil-like  appear- 
ance, while  in  Entamoeba  histolytica  they  are  much 
longer  and  finger-like  in  shape  and  present  a  dense, 
glassy  appearance.  In  some  of  the  free-living  forms 
the  pseudopodia  are  always  spinose  in  shape,  differ- 
ing entirely  in  appearance  from  those  of  the  parasitic 
amoebae.  I  have  never  observed  spinose  pseudopodia 
in  any  of  the  parasitic  amoebse  of  man. 


MORPHOLOGY  AND  BIOLOGY.  23 

The  exact  mechanism  of  the  origin  of  the  pseudo- 
podia  has  been  studied  by  many  observers,  and 
numerous  theories  have  been  brought  forward  to  ex- 
plain this  interesting  phenomenon.  However,  it  may 
be  stated  that  we  are  yet  ignorant  of  the  real 
mechanism  of  pseudopodia  formation. 

In  one  genus  of  amoebae,  i.e.,  Paramceba,  delicate 
ftagella  have  been  observed  during  certain  stages  of 
development.  Organisms  belonging  to  this  genus 
possess  both  an  amoeboid  and  flagellate  cycle  of  de- 
velopment. During  the  amoeboid  cycle  they  move 
about  by  means  of  pseudopodia,  while  during  the 
flagellate  cycle  locomotion  is  accomplished  by  means 
of  a  flagellum  wrhich  apparently  takes  its  origin  from 
some  portion  of  the  periphery  of  the  organism. 

METHODS  OF  REPRODUCTION. — Although  these 
organisms,  from  a  morphological  standpoint,  illus- 
trate the  most  simple  form  of  animal  life,  they  possess 
several  methods  of  reproduction,  some  of  them  of 
great  complexity.  These  methods  are:  simple 
division,  schizogony,  gemmation,  and  reproduction 
within  a  cyst.  The  first  three  methods  occur  during 
the  vegetative  stage  of  existence,  the  latter,  only  when 
conditions  arise  which  are  unfavorable  to  such  ex- 
istence. Schizogony  and  reproduction  within  a  cyst 
are  confined  to  certain  species,  and  gemmation  ap- 
pears to  be  peculiar  to  Entamceba  Mstolytica,  but 
simple  division  occurs  in  all  species. 


24  PARASITIC  AMGEB^E  OF  MAN. 

Simple  Division. — This  method  of  reproduction 
is  asexual  in  nature  and  consists  of  a  primary  division 
of  the  nucleus,  which  may  be  either  mitotic  or  amitotic 
in  character,  followed  by  the  division  of  the  cyto- 
plasm, two  amoeba*  being  thus  produced.  This 
method  of  reproduction  occurs  in  all  species  of 
amoebae,  so  far  as  is  known. 

Schizogony. — This  method  of  reproduction  is  also 
asexual  in  nature  and  is  accomplished  by  the  division 
of  the  karyosome  by  primitive  mitosis,  after  which  the 
nuclear  chromatin  becomes  collected  around  the 
nuclear  membrane  while  the  cytoplasm  rids  itself 
of  all  foreign  material.  When  this  is  completed, 
the  chromatin  divides  into  from  four  to  eight  little 
masses,  the  nuclear  membrane  ruptures  and  the 
chromatin  masses  become  free  in  the  endoplasm. 
Finally,  the  cytoplasm  divides  into  as  many  parts 
as  there  are  chromatin  masses,  and  a  number  of 
daughter  amoebae  are  thus  formed.  This  process  will 
be  described  in  detail  in  the  consideration  of  En- 
tamceba  coli,  and  Entamaeba  tetragena. 

Gemmation. — This  process  of  reproduction,  as 
observed  in  amoebae,  was  first  described  by  Schaudinn 
as  occurring  in  Entamceba  histolytica.  In  consists 
in  amitotic  division  of  the  nucleus,  the  chromatin 
being  dispersed  in  the  cytoplasm,  and  collecting  at 
the  periphery  of  the  organism  where  it  is  budded  off, 


>'    2 


FIG.  I. —  Diagram  illustrating  the  life-cycle  of  Entamoeba  coli.  (After 
Hartmann.)  1,  young  amoeba;  2,  adult  amoeba;  2a,  reproduction  by  simple 
division;  3,  the  division  of  the  chromatin  of  the  nucleus  into  eight  groups 
collected  upon  the  surface  of  the  nuclear  membrane;  4,  complete  nuclear 
division;  5,  schizogony,  or  division  of  the  amoeba  into  eight  daughter 
amoebae;  6,  commencement  of  cyst  formation;  7,  primary  division  of  the 
nucleus  within  the  cyst;  8,  incomplete  cell-division  and  formation  of  chro- 
mides;  9,  formation  by  the  chromides  of  two  pairing  nuclei;  10,  formation 
of  two  reduced  nuclei  by  each  pairing  nucleus;  11,  division  of  each  of  the 
reduced  nuclei  into  a  free  nucleus  (male)  and  a  stationary  nucleus  (female); 
12,  two  nuclear  stage  formed  by  the  merging  of  one  free  and  one  stationary 
nucleus;  13,  adult  cyst  containing  eight  daughter-nuclei  formed  by  the 
repeated  division  of  the  new  nucleus;  14,  the  adult  cyst  after  reaching  the 
intestine  of  the  host,  liberating  the  eight  young  amoebae  formed  during 
sporogony. 


MORPHOLOGY  AND  BIOLOGY.  25 

each  mass  of  chromatin  being  surrounded  by  a  little 
cytoplasm,  thus  forming  a  young  amoeba.  During 
this  process  some  of  the  chromatin  is  extruded  from 
the  parasite  while  some  remains  as  a  residual  mass  or 
masses. 

Reproduction  Within  a  Cyst. — When  conditions 
are  unfavorable  for  the  vegetative  existence  of  amoebae 
they  undergo  encystment  and  reproduction  occurs 
within  the  cysts.  The  method  of  reproduction  within 
a  cyst  varies  in  different  species  and  occurs  in  two 
different  ways:  either  by  the  primitive  mitosis  of 
the  nucleus,  followed  by  autogamous  fertilization ;  or 
by  the  nuclear  chromatin  becoming  dispersed  through- 
out the  cytoplasm  followed  by  the  formation  of 
minute  buds  containing  chromidia  which  are  separated 
from  the  parent  body  and  form  spores  by  the  secre- 
tion of  a  resisting  membrane.  In  this  condition  they 
are  incapable  of  undergoing  further  development 
until  surrounded  by  conditions  favorable  to  vegeta- 
tive life.  These  methods  of  reproduction  will  be 
considered  more  fully  in  the  discussion  of  individual 
amoeba?. 

Conjugation. — It  has  been  demonstrated  that  in 
certain  species  of  amoebae  conjugation  between  two 
individuals  not  infrequently  occurs.  I  have  observed 
it  repeatedly  in  Entamceba  coli,  and  rarely  in 
Entamceba  histolytica  and  Entamceba  tetragena. 


26  PARASITIC  AMOEBA  OF  MAN. 

It  is  uncertain  just  what  occurs  during  the  conjuga- 
tion of  organisms  of  this  class,  but  it  is  probable  that 
it  leads  to  a  rejuvenescence  of  the  vital  processes 
rather  than  that  it  is  a  process  of  fertilization. 

RESISTANCE  TO  PHYSICAL  CONDITIONS  AND  CHEM- 
ICAL AGENCIES. — Some  species  of  free-living  amoebae 
are  able  to  withstand  a  considerable  degree  of  heat 
and  cold,  but  the  parasitic  amoebae  of  man  become 
motionless,  as  a  rule,  after  exposure  for  an  hour  to 
temperatures  below  75°  F.  It  has  been  suggested  by 
some  authorities  that  the  species  living  in  man  can 
be  best  distinguished  from  free-living  forms  by  their 
lesser  resistance  to  heat  and  cold,  the  free-living 
forms  remaining  motile  when  exposed  to  great  varia- 
tions in  temperature,  while  the  parasitic  amoebae  are 
only  motile  at  a  temperature  near  that  of  the  human 
body.  The  writer  does  not  believe  that  this  method 
of  differentiation  is  of  much  value,  as  he  has  fre- 
quently observed  amoebae,  belonging  to  the  genus 
Entamceba,  moving  about  on  a  microscopic  slide  at 
a  temperature  far  below  that  to  which  they  are 
accustomed  in  the  human  intestine.  Harris  and  Mus- 
grave  have  shown  that  a  temperature  of  0°  F.  is 
not  sufficient  to  kill  some  amoebae,  but  it  should  be 
remembered  that  only  the  encysted  forms  are  capable 
of  withstanding  such  low  temperatures. 

As  regards  the  resistance  of  these  organisms  to 


MORPHOLOGY  AND  BIOLOGY.  27 

chemical  agencies  it  may  be  stated  that  a  large  number 
of  such  substances  are  capable  of  destroying  them.    A 
1  to  300  solution  of  quinine  sulphate  will  kill  the 
amoebae  of  man  within  a  few  minutes,  while  weak 
solutions    of    hydrogen    dioxide,    permanganate    of 
potassium,  sulphate  of  copper,  nitrate  of  silver,  argy- 
rol,  and  dilute  acids  are  efficient  agents  in  the  de- 
struction of  this  class  of  organisms.    Unfortunately, 
the  parasitic  amoebae  are  not  as  easily  destroyed  when 
present  in  the  intestine  as  they  are  upon  a  microscopic 
slide,  as  the  pathogenic  forms  penetrate  deep  into 
the  tissues  where  chemical  solutions  cannot  reach 
them.     Our  knowledge  is  incomplete  as  regards  the 
effect  of  chemicals  upon  the  amoebae  of  man,  as  most 
of  the  experimental  work  has  been  done  with  cultures 
of  free-living  species.    In  a  recent  paper  Vedder  gives 
in  detail  some  very  important  experiments  in  which 
he  found  that  ipecac,  not  de-emetized,  killed  a  cul- 
tural amoeba  in  dilutions  as  high  as  1-50,000  in  24 
hours;  emetin,    1-100,000;  quinine,  1-200,000;  and 
silver  nitrate  1-300,000.     How  far  his  results  apply 
to  the  parasitic  amoebae  of  man  is  undetermined. 

Many  other  chemicals,  strong  electrical  currents, 
and  the  Roentgen  rays  are  capable  of  destroying 
amoebae,  but  none  of  these  agents  have  proven  of 
much  service  in  the  treatment  of  amoebic  dysentery. 


III. 

CLASSIFICATION  AND  NOMENCLATURE. 

FROM  the  time  of  the  first  description  of  a  para- 
sitic amoeba  of  man  the  classification  and  nomen- 
clature of  these  organisms  has  occasioned  much  con- 
fusion and  difficulty.  This  is  due  to  the  great 
resemblance  in  the  general  morphology  of  the  para- 
sites belonging  to  the  various  species  and  to  the 
difficulty  and  labor  of  studying  their  life  cycle. 
While  for  years  all  authorities  have  agreed  that  these 
organisms  belong  to  the  Protozoa,  almost  every 
writer,  until  recently,  has  differed  in  his  classification 
or  in  his  conception  of  the  biological  history  of  the 
amoeba?  associated  with  dysentery,  and  it  was  not 
until  Schaudinn's  observations  were  published  that 
a  really  satisfactory  classification  of  these  parasites 
was  possible. 

However,  it  is  probable  that  the  present  accepted 
classification  may  be  found  more  or  less  erroneous 
as  our  knowledge  increases,  and  it  should  be  under- 
stood that  in  any  of  the  amaebce  in  which  the  life 
cycle  has  not  been  thoroughly  worked  out,  classifica- 
tion can  only  be  provisional.  There  are  a  few  well- 
studied  species  occurring  in  man  in  which  the  present 

28 


CLASSIFICATION  AND  NOMENCLATURE.      29 

classification  may  be  said  to  be  final,  but  other  species 
have  been  described  and  classified  which  are  still  in 
doubt  and  in  which  the  present  classification  may  or 
may  not  be  correct. 

The  Amcebina  are  divided  by  zoologists  into  the 
Reticulosa,  having  filamentous  pseudopodia ;  and  the 
Lobosa,  having  lobose  pseudopodia;  the  latter  are 
divided  into  the  Gymnamceba,  in  which  the  proto- 
plasm is  naked;  and  the  Testacea,  in  which  the 
protoplasm  is  surrounded  by  a  shell. 

The  parasitic  amoeba?  of  man  belong  to  the 
Gymnamceba,  or  naked  amoebae  having  lobose  pseudo- 
podia, and  are  placed  in  the  genus  Entamoeba  created 
by  Casagrandi  and  Barbagallo.  All  free-living  forms 
are  placed  in  the  genus  Amoeba  and  are  differentiated 
from  the  members  of  the  genus  Entamoeba  by  the 
presence  of  a  contractile  vacuole  and  other  morpholog- 
ical characteristics,  and  their  inability  to  exist  as 
parasites  within  man. 

In  addition  to  the  two  genera  mentioned,  we 
must  include  the  genus  Paramceba,  which  contains  a 
marine  amoeba,  Paramceba  eilhardi,  and  the  parasitic 
form  known  as  Paramceba  hominis. 

The  absence  of  amoebae  in  numerous  typical  cases 
of  dysentery,  and  their  presence  in  health  and  dis- 
eases other  than  dysentery,  has  gradually  led  to  the 
grouping  of  students  of  the  subject  into  three  schools, 


30  PARASITIC  AMCEB.E  OF  MAN. 

i.e.,  those  believing  that  amoebae  are  always  harmless 
commensals,  more  numerous,  it  is  true,  in  the  feces 
of  dysentery  patients  because  of  the  more  favorable 
environment ;  those  believing  that  all  amoebae  may  be 
pathogenic  if  suitable  conditions  be  present;  and 
those  believing  that  both  harmless  and  pathogenic 
species  may  be  present  in  the  intestine  of  man. 

Prior  to  Schaudinn's  work  several  investigators 
had  endeavored  to  establish  a  classification  of  these 
parasites  based  upon  morphological  differences,  but 
without  success  so  far  as  the  general  acceptance  of 
any  one  classification. 

In  1893,  Quincke  and  Roos,  as  the  result  of  their 
studies,  divided  the  amoebae  of  man  into  three  species, 
as  follows: 

1.  Amoeba    intestini    vulgaris,    40    microns    in 
diameter,  with  large  granules,  which  was  pathogenic 
for  neither  man  nor  cats. 

2.  Amoeba  coli  mitis,  similar  in  size  and  appear- 
ance to  the  preceding,  but  which  was  pathogenic  for 
man  alone. 

3.  Amoeba  coli   (Loesch),  about  25  microns  in 
diameter,  with  a  finely  granular  endoplasm,  which 
produced  dysentery  in  both  man  and  cats. 

This  classification,  which  was  based  largely  upon 
the  results  of  animal  experiments,  was  not  conclusive, 
as  from  the  description  of  the  organisms  it  was  evi- 


CLASSIFICATION  AND  NOMENCLATURE.      31 

dent  that  the  authors  were  dealing  with  mixed  in- 
fections with  both  harmless  and  pathogenic  amoebae. 
In  addition,  the  names  given  to  the  first  two  species 
cannot  stand,  as  they  are  not  in  accordance  with  the 
binominal  system  adopted  in  nomenclature. 

In  1894  Kruse  and  Pasquale  distinguished  four 
varieties  of  amoebae  based  entirely  upon  morphological 
characteristics:  (1)  a  form  presenting  a  very  re- 
fractive protoplasm,  found  in  normal  feces;  (2)  a 
form  showing  irregular  and  small  granules;  (3)  a 
form  in  which  the  endoplasm  consisted  largely  of 
vacuoles;  (4)  a  form  in  which  the  protoplasm  was 
filled  with  foreign  bodies.  The  two  latter  forms 
were  found  only  in  dysenteric  feces. 

It  will  at  once  be  seen  that  the  differences  in  these 
forms  are  so  slight  as  to  be  of  no  scientific  value  as 
a  basis  of  classification,  and  the  fact  that  these 
authors  described  the  form  found  in  normal  feces  as 
being  very  refractive,  is  evidence  that  they  confused 
the  pathogenic  and  harmless  species,  although  they 
recognized  a  pathogenic  amoeba,  Amoeba  dy sentence, 
following  Councilman  and  Lafleur  who,  in  1891,  ob- 
jected to  Amoeba  coli,  as  a  name  for  the  amoeba 
causing  dysentery,  and  suggested  the  name  "Amoeba 
dysenteric?  "  The  latter  authors,  while  not  attempt- 
ing to  differentiate  species,  expressed  it  as  their 
opinion  that  under  certain  conditions  both  harmless 


32  PARASITIC  AMCEB.E  OF  MAN. 

and  pathogenic  amoebae  may  inhabit  the  intestine  of 
man. 

Celli  and  Fiocca,  in  1895,  described  no  less  than 
six  species  of  amoebae  infesting  the  intestine  of  man 
and  named  these  organisms  as  follows:  (1)  Amoeba 
spinosa;  (2)  Amoeba  vermicularis;  (3)  Amoeba 
diaphana;  (4)  Amoeba  reticularis;  (5)  Amoeba 
lobosa,  variety  guttata;  (6)  Amoeba  lobosa,  variety 
oblonga.  The  names  sufficiently  describe  the  differ- 
ential characteristics  of  these  organisms,  but  they  all 
differed  in  size,  one  species,  A.  diaphana,  measuring 
only  0.5  to  2  microns  in  diameter.  To  one  who  has 
studied  these  organisms  the  description  of  an  amoeba 
measuring  but  0.5  micron  in  diameter  may  well  be 
viewed  with  suspicion,  when  we  consider  that  such  an 
organism  would  be  but  little  larger  than  the  smallest 
micrococcus.  The  largest  amoebae  described  by  Celli 
and  Fiocca  did  not  exceed  10  microns  in  diameter, 
which  proves  that  these  authors  were  not  dealing 
with  true  dysentery  amoebae  and  it  is  more  than  prob- 
able that  many  of  the  species  described  by  them  were 
really  stages  in  the  development  of  the  intestinal 
flagellates. 

Casagrandi  and  Barbagallo  were  the  first  in- 
vestigators to  accurately  describe  the  species  of 
amoeba  occurring  in  the  feces  of  healthy  individuals 
and  now  known  as  Entamoeba  coli.  They  established 


CLASSIFICATION  AND  NOMENCLATURE.      35 

for  this  amoeba  the  genus  Entamceba  because  they 
considered  that  these  organisms  differed  both  in 
morphology  and  life  history  from  the  fresh  water 
amoebae.  They,  described  very  minutely  the  mor- 
phology and  method  of  reproduction  of  this  species 
and  their  investigations  directed  the  attention  of 
other  workers  to  the  occurrence  of  amoebae  in  health 
and  in  diseases  other  than  dysentery.  However,  they 
were  not  successful  in  differentiating  the  pathogenic 
and  harmless  amoebae. 

In  1900,  Strong  and  Musgrave  recognized  two 
species  of  amoebae  occurring  in  their  patients  in 
Manila,  and  state  that  with  the  harmless  amoeba, 
which  they  call  Amoeba  coli,  they  were  never  able  to 
produce  dysentery  in  cats ;  while  with  the  pathogenic 
amoeba,  Amoeba  dy sentence,  they  had  no  difficulty 
in  producing  the  disease  in  cats  by  the  injection  of 
f eces  or  the  contents  of  liver  abscesses  containing  the 
living  parasites. 

While,  according  to  Schaudinn,  the  first  in- 
vestigator to  clearly  identify  and  describe  a  patho- 
genic and  non-pathogenic  species  of  amoeba  in  man 
was  Jiirgens,  in  1902,  I  believe  that  to  Strong  and 
Musgrave  really  belongs  the  credit  of  making  this 
observation,  as  their  work  was  published  in  1900,  while 
that  of  Jiirgens  did  not  appear  until  1902.  There  is 
but  little  doubt  that  Strong  and  Musgrave  actually 


34  PARASITIC  AMCEB^E  OF  MAN. 

differentiated  the  two  amoebae  now  known  as  En- 
tamceba coli  and  Entamceba  histolytica,  but  unfortu- 
nately they  did  not  adopt  the  name  Entamceba  as  a 
generic  term. 

In  1903,  Schaudinn,  following  the  work  of 
Jiirgens,  definitely  proved  that  two  distinct  species 
of  amoebae  infested  the  intestine  of  man.  His  work 
rested  not  only  upon  fiiff  erences  in  the  morphology 
of  the  two  parasites,  but  upon  their  entirely  distinct 
methods  of  reproduction,  which  he  studied  very  thor- 
oughly. To  the  amoeba  occurring  in  the  feces  of 
normal  individuals,  or  in  those  suffering  from  other 
diseases  than  dysentery,  he  gave  the  name  Entamceba 
coli,  while  to  those  occurring  in  dysenteric  feces  and 
causing  that  disease,  he  gave  the  name  Entamceba 
histolytica. 

Stiles  has  written  an  exhaustive  summary  regard- 
ing the  nomenclature  of  the  amoebae  of  man,  in  which 
he  clearly  states  the  nomenclatural  situation,  and  con- 
cludes that  for  those  who  believe  that  there  is  but 
one  species  of  amoeba  infecting  man  and  that  it  is 
not  congeneric  with  fresh  water  amoebae,  the  correct 
name  to  use  is  Entamceba  coli,,  not  Amoeba  coli,,  for, 
as  shown  by  Casagrandi  and  Barbagallo,  the  amoebae 
of  the  human  intestine  differ  from  the  fresh  water 
amoebae,  to  which  the  generic  term  "  Chaos,"  later 
amended  by  Ehrenberg  to  "  Amoeba,"  was  originally 
given;  while  for  those  who  believe  in  a  harmless  and 


CLASSIFICATION  AND  NOMENCLATURE.      35 

a  pathogenic  species,  the  correct  zoological  names  are 
Entamceba  coli  and  Entamoeba  Mstolytica  respec- 
tively. 

In  1905,  I  suggested  that  as  the  name  Amoebce 
dysenteries  had  been  given  the  pathogenic  species  by 
Councilman  and  Lafleur,  the  name  Mstolytica  should 
give  way  to  dysenterice,  but  as  Stiles  has  conclusively 
shown  that  the  latter  is  merely  a  synonym  of  coli,  it 
follows  that  it  cannot  be  used  to  designate  the  patho- 
genic amoeba,  and  therefore  we  must  accept  Schau- 
dinn's  name  Entamoeba  Mstolytica  for  one  of  the 
species  of  amoebae  causing  amoebic  dysentery. 

At  the  present  time  Schaudinn's  classification  has 
been  accepted  by  most  medical  investigators  and 
writers,  and  by  all  zoologists  with  whose  work  I  am 
acquainted.  Musgrave  and  Clegg,  almost  alone  of 
those  who  have  had  an  extensive  experience  with 
amoebic  dysentery,  still  decline  to  accept  Schaudinn's 
classification,  although  they  state  "we  do  not  at  all 
question  the  multiplicity  of  both  genera  and  species 
of  amoebae,  both  within  and  without  the  intestine  of 
man."  They  also  adhere  to  the  name  Amoeba  coUf 
although,  as  Stiles  has  shown,  the  proper  generic 
term  for  the  parasitic  amoebae  of  man  is  Entamoeba. 

The  exact  zoological  position  of  the  parasitic 
amoebae  of  man,  together  with  their  specific  names, 
may  be  tabulated  as  follows: 


36  PARASITIC  AMOEBA  OF  MAN. 

Phylum,  Protozoa. 

Sub-phylum,  Sarcodina. 
Class,  Rhizopoda. 
Sub-class,  Amcebina. 
Order,  Gymnamcebida. 

.Genus,  Entamceba,  Casagrandi  and  Barbagallo, 
1895. 

Species,  Entamceba  coll,   Losch,    1875,    emend 

Schaudinn,  1903. 
Entamceba      liistolytica,      Schaudinn, 

1903. 

Entamceba  buccalis,  Prowazek,  1904. 
Entamceba  tetragena,  Viereck,  1907. 
Entamceba     phagocytoides,      Gaudu- 

cheau,  1908. 

Entamceba  tropicalis,  Lesage,  1908. 
Entamceba  minuta,  Elmassian,  1909. 
Entamceba  nipponica,  Koidzumi,  1909. 
Entamceba  kartulisi,  Doflein,  1901. 
Genus,  Paramoeba,  Schaudinn,  1896. 
Species,  Paramceba  hominis,  Craig,  1906. 

There  are  a  number  of  other  species  of  question- 
able value  and  it  is  possible  that  some  of  the  species 
given  above  may  have  to  be  relinquished  upon  further 
study.  Students  of  these  parasites  are  becoming 


CLASSIFICATION  AND  NOMENCLATURE.      37 

more  and  more  convinced  that  species  differentiation 
should  rest  chiefly  upon  differences  in  the  life  cycle 
rather  than  upon  mere  morphological  variations,  and 
the  writer  believes  that  a  species  should  not  be  de- 
scribed until  at  least  the  larger  portion  of  its  life 
cycle  has  been  demonstrated. 

The  species  which  are  still  of  uncertain  value  are 
the  following: 

Genus,  Entamoeba. 

Species,  Entamoeba  undulans,  Castellani,  1905. 

Amoeba  pulmonalis,  Artault,  1900. 

Amoeba  urogewtalis,  Baelz,  1903. 

Amoeba  miurai,  Ijima,  1898. 

Amoeba  gingivalis,  Gros,  1848. 

Amoeba  dentalis,  Grassi,  1879. 


TECHNIQUE. 

THE  parasitic  amoebae  of  man  may  be  studied  in 
the  living  condition,  in  stained  smears  of  material 
containing  them,  and  in  stained  sections  of  tissue.  Each 
of  these  methods  has  its  advantages,  but  for  diagnosis 
and  for  the  study  of  the  vital  activities  of  these 
parasites  the  examination  of  fresh  material  is  the 
most  satisfactory.  It  is  never  necessary  for  diag- 
nostic purposes  to  stain  amoebae  nor  is  it  essential  for 
the  differentiation  of  species.  It  is  very  difficult  to 
stain  these  organisms  and  even  with  the  utmost  care 
it  will  often  be  found  that  scores  of  preparations 
will  have  to  be  examined  before  a  satisfactory  one 
is  obtained.  As  fixing  and  staining  the  organisms 
often  cause  them  to  assume  appearances  very  unlike 
those  observed  in  living  specimens,  it  is  my  belief  that 
stained  preparations  should  never  be  depended  upon 
for  diagnosis  except  in  the  hands  of  an  expert. 

However,  the  use  of  stained  smears  is  necessary 
in  following  out  the  nuclear  changes  during  the  life 
cycle  of  these  organisms,  for  while  in  the  living  speci- 
mens such  changes  may  be  observed,  they  are  much 
more  definitely  marked  in  the  stained  preparations. 

38 


TECHNIQUE.  39 

The  typical  staining  reaction  of  the  chromatin  makes 
it  easy  to  follow  the  changes  occurring  in  the  nucleus 
during  the  various  reproductive  processes.  For  this 
purpose  no  other  method  gives  as  good  results  as 
the  use  of  carefully  stained  specimens. 

The  great  advantage  of  the  use  of  stained  sections 
of  tissue  lies  in  the  knowledge  which  they  furnish  us 
regarding  the  exact  relation  of  amoebae  to  the  patho- 
logical conditions  present  in  the  tissues  examined. 
Thus  in  sections  of  the  intestine  from  patients  dying 
of  amoebic  dysentery  the  stained  sections  demonstrate 
the  undoubted  etiological  relationship  of  the  amoebae 
to  certain  of  the  lesions  present,  and  this  is  also  true 
of  sections  of  the  liver  in  which  abscesses  due  to  these 
organisms  are  found.  In  no  other  way  can  we  as 
well  demonstrate  the  relationship  of  amoebae  to  the 
lesions  which  they  produce  in  man. 

THE  EXAMINATION  OF  LIVING  AMGEB^E. — A  very 
small  portion  of  a  freshly  passed  stool  should  be 
placed  upon  a  microscopic  slide  and  covered  with  a 
cover  glass,  gentle  pressure  being  used  to  spread  the 
specimen.  The  material  selected  for  examination 
should  preferably  be  a  drop  of  the  liquid  portion 
of  the  stool  rather  than  solid  particles.  It  is  always 
well  to  give  a  saline  cathartic  before  making  an 
examination  as  this  tends  to  wash  the  amoebae  from 
the  intestinal  walls.  If  present,  a  particle  of  mucus 


40  PARASITIC  AMOEBAE  OF  MAN. 

or  any  blood-stained  material  should  be  examined  as 
well.  Most  stools  from  amoebic  dysentery  cases  con- 
tain gelatinous  material  which  frequently  contains 
numerous  amoebae,  and  such  material  should  always 
be  fully  examined. 

The  feces  should  be  examined  as  quickly  as  pos- 
sible after  they  have  been  passed  as  the  amoebae  are 
much  more  easily  recognized  when  they  are  motile. 
No  disinfectant  should  be  used  in  stools  which  are 
to  be  examined  for  amoebae,  nor  should  urine  be 
mixed  with  the  stools.  In  temperate  regions,  espe- 
cially in  the  winter,  the  receptacle  used  in  collecting 
the  specimen  should  be  warmed,  but  care  should  be 
taken  if  water  is  used  for  this  purpose,  that  it  be 
boiled,  as  otherwise  water  amoebae  might  be  mistaken 
for  parasitic  amoebae,  having  reached  the  feces  in  this 
manner. 

Unless  the  reproductive  cycle  of  an  organism  is 
to  be  studied  the  warm-stage  is  not  necessary,  but  it 
should  always  be  used  in  research  work  upon  these 
parasites,  as  in  no  other  way  can  the  various  stages 
in  growth  and  reproduction  be  thoroughly  studied. 
When  extensive  studies  of  the  vital  activities  of 
amoebae  are  to  be  undertaken,  an  incubator,  designed 
to  contain  the  microscope,  will  be  found  to  be  a  great 
convenience. 

Although  it  is  generally  stated  that  entamoebae 


TECHNIQUE. 


41 


become  motionless  at  room  temperature  after  an  hour 
or  so,  it  is  not  unusual  to  observe  motility  in  these 
parasites  in  specimens  of  feces  which  have  been  col- 
lected for  several  hours.  If  the  amoebae  are  not 
motile  the  gentle  warming  of  the  slide  will  often 
restore  motility,  provided  degeneration  of  the  para- 
sites has  not  occurred.  Even  when  the  amoebae  are 
motionless  it  is  not  difficult,  for  one  accustomed  to 
observing  them,  to  distinguish  them  from  other  bodies 
occurring  in  the  feces.  Many  observers  claim  that 
a  diagnosis  of  the  presence  of  amoebae  in  feces  should 
never  be  made  unless  the  organisms  be  motile. 
While  this  is  valuable  advice  for  the  novice  in  this 
line  of  work,  it  is  certainly  true  that  by  one  who  has 
studied  these  organisms,  they  can  be  easily  recog- 
nized when  motionless,  provided  the  feces  has  not 
been  kept  so  long  as  to  have  led  to  degenerative 
changes  in  the  amoebae. 

In  making  fresh  preparations  it  is  always  well 
to  dilute  a  loopful  of  the  stool  with  normal  salt 
solution  or  distilled  water.  One  of  the  most  frequent 
mistakes  made  in  examining  such  preparations  is  the 
use  of  too  thick  a  preparation.  One  should  not  be 
satisfied  with  the  examination  of  a  single  slide,  but 
should  thoroughly  examine  at  least  six  or  eight 
preparations  before  a  negative  result  is  reported. 

The  hanging  drop  method  is  often  valuable  in 


42  PARASITIC  AMOEBA  OF  MAN. 

the  examination  of  these  organisms,  a  small  drop  of 
the  stool  being  placed  in  the  centre  of  a  cover  glass 
which  is  then  inverted  upon  a  hollow  ground  slide 
and  ringed  with  vaseline.  If  a  film  preparation  is 
used  it  should  always  be  ringed  with  vaseline,  for 
unless  this  is  done  evaporation  occurs  and  the  prepa- 
ration becomes  useless. 

The  preparation  should  be  examined  with  a  one- 
sixth  inch  lens  and  a  one-  or  two-inch  eyepiece.  For 
the  finer  details  regarding  the  structure  of  the  cyto- 
plasm and  the  nucleus,  as  well  as  the  reproductive 
changes,  it  is  necessary  to  use  the  one-twelfth  inch 
oil  immersion  objective. 

Neutral  Red. — The  use  of  a  solution  of  1/10,000 
of  neutral  red  is  often  of  great  service  in  those  cases 
in  which  the  amoebae  are  few  in  number  and  for  the 
study  of  structural  details.  This  solution  is  very 
quickly  absorbed  by  the  amoeba?,  coloring  them  pink 
or  red,  and  does  not  interfere  with  their  movements 
if  it  is  not  used  in  too  strong  a  dilution.  It  is  a 
most  useful  method  in  distinguishing  between  para- 
sitic amoeba?  and  leucocytes,  as  the  latter  do  not  stain 
with  this  substance.  In  specimens  in  which  the 
amoebae  are  in  scant  numbers  they  are  easily  dis- 
tinguished by  the  reddish  color  given  them  by  the 
neutral  red,  as  other  cells  occurring  in  feces  are  not 
colored  distinctly  by  this  dye.  The  dilution  should 
be  made  with  normal  salt  solution. 


TECHNIQUE.  43 

METHODS  OF  STAINING  MATERIAL  CONTAINING 
AMCEB^E. — Owing  to  the  very  delicate  structure  of 
amoebae  any  method  of  fixation  and  staining  which 
may  be  adopted  undoubtedly  causes  some  change  in 
size  and  morphology  and  one's  object  should  be  to 
use  methods  which  will  obviate,  as  far  as  possible,  such 
artificial  changes.  For  the  best  results  in  the  study 
of  the  morphology  of  the  nucleus  of  amoebse,  as  well 
as  other  Protozoa,  and  more  especially  the  changes 
occurring  in  this  structure  during  multiplication,  fixa- 
tion of  the  wet  specimen  with  osmic  acid  vapor  or 
sublimate  alcohol,  followed  by  staining  while  the 
specimen  is  still  wet,  is  essential,  although  air-dried 
specimens,  which  are  afterward  stained,  are  sufficient 
for  ordinary  work.  I  was  able  to  confirm  all  the 
essential  details  of  Schaudinn's  work  upon  Entamoeba 
histolytica  and  Entamoeba  coU  in  air-dried  smears  of 
material  containing  these  parasites,  stained  with 
Wright's  modification  of  the  Romanowsky  stain, 
although  only  after  months  of  study  and  the  examina- 
tion of  many  thousands  of  preparations.  Had  the 
wet  fixing  and  staining  methods  been  used  at  that 
time  much  labor  would  have  been  saved  and  the  mitotic 
division  of  the  nucleus  would  have  been  demonstrated. 
In  making  stained  preparations  it  is  first  necessary  to 
fix  upon  a  slide  or  cover  glass  the  material  to  be  ex- 
amined. For  this  purpose  numerous  fixing  mixtures 


44  PARASITIC  AMOEBAE  OF  MAN. 

have  been  elaborated.  Among  the  most  important 
are  the  following: 

Osmic  Acid. — This  mixture  consists  of  two  parts 
of  osmic  acid  in  100  parts  of  a  1  per  cent,  chromic 
acid  solution,  or  a  4  per  cent,  solution  of  osmic  acid  to 
which  a  certain  amount  of  glacial  acetic  acid  is  added 
at  the  time  of  fixation.  ( 1  drop  of  glacial  acetic  acid 
to  20  drops  of  the  4  per  cent,  solution  of  osmic  acid.) 

It  should  be  remembered  that  osmic  acid  is  a 
strong  irritant  of  the  mucous  membranes  and  care 
should  be  taken  not  to  inhale  the  fumes  or  allow  them 
to  come  in  contact  with  the  eyes.  The  sealed  tube 
containing  the  acid  should  be  broken  in  a  glass  stop- 
pered bottle  containing  the  desired  amount  of  water 
or  other  solution. 

Absolute  Alcohol. — When  dry  cover  glass  prepa- 
rations are  to  be  examined  they  may  be  fixed  by 
immersing  them  in  absolute  alcohol  for  from  2  to  5 
minutes. 

Alcoholic  Solution  of  Mercuric  Chloride. — The 
mercuric  chloride  used  in  this  mixture  is  obtained  by 
dissolving  perchloride  of  mercury  in  boiling  normal 
saline  solution  in  such  proportions  that  a  few  crystals 
of  perchloride  of  mercury  are  deposited  in  the  vessel 
after  boiling.  In  other  words,  a  saturated  solution 
of  perchloride  of  mercury  in  boiling  saline  solution 
is  to  be  obtained. 


TECHNIQUE.  45 

The  mixture  is  made  by  adding  one  part  of  abso- 
lute alcohol  to  2  parts  of  the  saturated  solution  of 
mercuric  chloride  and  it  should  be  used  warm,  the 
preparations  being  immersed  for  from  2  to  5  minutes. 
They  are  then  washed  in  50  per  cent,  alcohol,  and 
70  per  cent,  alcohol,  and  then  placed  in  70  per  cent, 
alcohol  to  which  enough  tincture  of  iodine  has  been 
added  to  give  a  port  wine  color.  They  are  allowed 
to  remain  in  this  mixture  until  they  turn  a  pale  yel- 
low, when  they  are  rinsed  in  70  per  cent,  alcohol, 
and  hardened  in  80  per  cent,  alcohol  for  at  least  a 
quarter  of  an  hour. 

Acetic  Acid  Solution  of  Picric  Acid  and  Mer- 
curial Sublimate. — This  mixture  consists  of  equal 
parts  of  a  saturated  solution  of  mercuric  chloride 
prepared  as  above ;  picric  acid,  1  per  cent,  in  distilled 
water;  and  a  J/£  to  1  per  cent,  solution  of  glacial 
acetic  acid.  This  method  is  most  useful  in  fixing 
portions  of  tissue  containing  amoeba?.  The  tissue 
should  be  left  in  the  mixture  over  night  and  then 
washed  in  50  per  cent,  and  70  per  cent,  alcohol. 

Sublimate  Acetic  Acid  Mixture. — This  mixture 
consists  of  95  parts  of  a  saturated  solution  of  sub- 
limate of  mercury  in  distilled  water  and  5  parts  of 
glacial  acetic  acid.  The  cover  glasses  containing  the 
material  to  be  examined  are  placed  directly  in  this 
mixture  while  wet  or  after  a  preliminary  fixation  with 
osmic  acid  vapor. 


46  PARASITIC  AMGEBJE  OF  MAN. 

Cover  glass  preparations  may  be  fixed  either  wet 
or  dry.  If  one  desires  to  study  the  minute  morphology 
of  amoebae  it  is  best  to  fix  the  preparations  wet, 
although  carefully  prepared  dry  specimens  often  give 
good  results,  and  the  writer  has  found  that  dry  prep- 
arations are  just  as  useful  for  ordinary  work.  If  the 
specimens  are  to  be  fixed  wet  the  following  procedure 
is  to  be  recommended: 

The  stool  containing  the  amoeba?  is  spread  in  as 
even  and  thin  a  layer  as  possible  upon  the  cover 
glass,  which  is  then  placed  with  the  material  down- 
wards in  a  dish  containing  the  fixing  solution.  While 
some  of  the  material  will  dissolve  in  the  solution,  it 
will  be  found  that  most  of  the  amoebae  are  retained 
upon  the  cover  glass.  After  fixing,  the  preparations 
are  rinsed  in  alcohol  (50  per  cent.)  and  hardened 
with  70  or  80  per  cent,  alcohol. 

A  quick  and  efficient  method  of  wet  fixation  is  by 
exposure  to  osmic  acid  vapor,  the  osmic  acid  solution 
already  described  being  used  for  this  purpose.  This 
method  is  most  useful  if  Giemsa's  or  Wright's  stain- 
ing methods  are  used  and  it  is  desired  to  avoid  artificial 
changes  in  the  structure  of  the  amoebae.  The  technique 
of  fixation  by  this  method  is  as  follows : 

A  cell,  slightly  smaller  in  diameter  than  the  cover 
glass  containing  the  material  to  be  examined,  and 
from  one-half  to  three-quarters  of  an  inch  deep,  is 


TECHNIQUE.  47 

hollowed  out  in  a  block  of  hard  paraffin.  The  osmic 
acid  solution  is  placed  within  this  cell,  the  latter  being 
filled  to  within  a  short  distance  of  the  surface.  The 
edge  of  the  paraffin  cell  is  then  smeared  with  vaseline 
and  the  cover  glass  then  placed  preparation-side 
downward  over  the  cell,  and  pressed  into  the  vaseline, 
thus  making  an  air-tight  junction.  After  exposure  to 
the  vapor  for  from  30  seconds  to  1  minute  the  prepa- 
ration is  at  once  placed  in  absolute  alcohol,  and  after- 
ward stained,  and  mounted  in  acid-free  Canada  bal- 
sam. At  no  stage  of  the  process  should  the  specimen 
be  allowed  to  dry  if  the  best  cytological  results  are 
desired.  The  osmic  acid  mixture  preferred  consists  of 
20  parts  of  a  4  per  cent,  osmic  acid  solution  and  1  part 
of  glacial  acetic  acid. 

Various  staining  methods  may  be  used  in  coloring 
the  parasitic  amoeba3  of  man.  Among  the  most  simple 
are  carbol-fuchsin,  methylene  blue,  gentian  violet  and 
hsematoxylin.  The  latter  is  most  valuable  in  demon- 
strating these  parasites  and  Delafield's  haematoxylin 
gives  the  best  results.  It  should  be  well  diluted  with 
distilled  water  and  the  preparations  should  be  left 
in  it  over  night,  after  which  they  are  washed  in  run- 
ning water,  and,  if  well  stained,  are  rinsed  in  50  per 
cent.,  70  per  cent.,  90  per  cent.,  and  absolute  alcohol, 
and  mounted  in  Canada  balsam,  which  is  free  from 
acid.  If  the  specimens  are  too  deeply  stained  they 


48  PARASITIC  AMCEB^  OF  MAN. 

may  be  decolorized  in  70  per  cent,  alcohol  contain- 
ing a  few  drops  of  hydrochloric  acid,  the  process 
being  watched  under  the  microscope  until  the  para- 
sites appear  a  pale  bluish  black.  They  are  then  placed 
in  a  solution  consisting  of  1  drop  of  ammonia  to  100 
c.c.  of  70  per  cent,  alcohol  until  the  color  turns  blue, 
when  they  are  rinsed  in  70  per  cent,  alcohol  and 
mounted.  If  desired,  the  cytoplasm  of  the  amoeba? 
may  be  stained  by  eosin  after  they  have  been  stained 
with  hgematoxylin  which,  of  course,  only  colors  the 
nucleus. 

If  carbol-f  uchsin,  methylene  blue,  or  gentian  violet 
is  used  for  staining,  the  fixed  preparations  are  placed 
in  the  staining  solution  for  a  few  moments,  washed 
in  70  per  cent,  alcohol,  and  mounted.  These  stains 
are  of  very  little  service  in  the  study  of  these  para- 
sites. 

The  Iron  Hcematoocylin  Method  of  Heidenhcdn. 
— This  is  a  most  valuable  method  for  studying  struc- 
tural details.  If  it  is  used  the  specimens  must  be 
fixed  wet  with  alcoholic  solution  of  mercuric  chloride 
or  with  osmic  acid  vapor.  The  following  solutions 
are  used  in  this  method: 

1.  A  2.5  per  cent,  watery  solution  of  iron-alum 
(Ferrous  ammonium  sulphate). 

2.  A    0.5    to    1    per    cent,    watery    solution    of 
hsematoxylin,  which  should  be  at  least  four  weeks  old. 


TECHNIQUE.  49 

Cover-glass  preparations  or  tissues,  previously 
rinsed  in  distilled  water  after  fixing,  should  be  placed 
in  solution  No.  1  over  night,  after  which  they  are 
rinsed  in  distilled  water  and  immersed  in  solution 
No.  2  for  from  6  to  24  hours  and  in  the  case  of 
sections  as  long  as  36  hours.  They  are  then  rinsed 
in  distilled  water  and  decolorized  in  solution  No.  1, 
this  process  being  controlled  by  observation  under  the 
microscope,  until  the  nucleus  is  well  differentiated, 
the  chromatin  staining  a  deep  blue-black  and  the 
cytoplasm  a  light  gray;  the  preparations  are  then 
washed  in  running  water  for  half  an  hour  and  rinsed 
in  50  per  cent.,  70  per  cent.,  90  per  cent,  and  absolute 
alcohol,  cleared  in  xylol,  and  mounted  in  Canada 
balsam. 

This  method  requires  considerable  experience  in 
order  to  obtain  the  best  results,  but  may  be  said  to 
be  the  best  method  we  possess  for  the  study  of  the 
structural  details  of  amoeba?. 

The  Giemsa  Stain. — Very  beautiful  specimens 
may  be  obtained  by  this  method.  Its  chief  objection 
is  that  the  method  of  preparation  is  so  complicated 
and  requires  so  much  care  that  it  can  only  be  used 
in  a  well  equipped  laboratory.  However,  a  reliable 
Giemsa  stain  may  be  obtained  from  Grubler.  The 
following  is  the  method  of  preparation  of  this  stain 
and  of  its  use  in  staining  amoebse : 


50  PARASITIC  AMCEBJE  OF  MAN. 

The  utmost  care  must  be  taken  that  everything 
used  in  preparing  this  stain,  and  using  it,  is  chemically 
clean,  and  that  none  of  the  dishes  or  implements 
used  are  moist  and  that  they  be  sterilized  before  use. 

The  stock  solution  consists  of  Azure  II-eosin,  3 
grams,  and  Azure  II,  0.8  gram.  These  are  dissolved, 
by  constantly  shaking,  in  250  c.c.  of  pure  glycerine 
at  a  temperature  of  60  degrees  C.  After  solution  is 
complete,  250  c.c.  of  absolute  methyl  alcohol  (Kaul- 
baum  I)  previously  heated  to  60  degrees  C.  is  added. 
This  mixture  is  well  shaken,  allowed  to  stand  at  room 
temperature  for  24  hours,  and  then  filtered  into  a 
chemically  clean,  sterilized,  air-tight  bottle.  During 
the  filtration  the  funnel  must  be  covered  in  order  to 
protect  the  hydroscopic  fluid  from  moisture  and  the 
bottle  containing  the  stock  solution  should  be  kept 
in  a  dark  place. 

The  method  of  using  this  stain  is  as  follows: 

Thin,  even  smears  of  the  material  containing  the 
amoebae  are  made  upon  cover  slips  and  fixed  while 
wet  in  osmic  acid  vapor  or  sublimate  alcohol,  and 
stained  with  the  following  solution: 

Stock  solution    10  drops. 

Distilled  water 10  c.c. 

1    per   cent  potass,  carb.   solution,    1    or   2   drops   to   each 
10  c.c.  of  the  above  mixture. 

This  staining  solution  is  allowed  to  act  for  from 
30  minutes  to  6  hours,  although  fairly  good  prepara- 


TECHNIQUE.  51 

tions  may  be  obtained  after  staining  for  from  10  to 
15  minutes.  The  stained  smears  are  finally  washed 
in  running  distilled  water,  the  excess  of  stain  removed 
with  alcohol,  cleared  with  xylol,  and  mounted  in  acid- 
free  Canada  balsam.  A  good  Giemsa  stain  may  be 
obtained  from  Grubler  and  is  used  in  the  proportion 
of  1  drop  of  the  stain  to  1  c.c.  of  distilled  water.  With 
this  wet-fixed  specimens  should  be  stained  for  from 
2  to  6  hours,  and  cleared  and  mounted  as  just  de- 
scribed. Fair  results  are  obtained  with  this  stain  in 
air-dried  preparations. 

Wright's  Method. — The  following  method  I  have 
found  very  satisfactory  in  staining  parasitic  amoebae 
and  with  it  I  have  been  able  to  follow  the  entire  cycle 
of  development  of  Entamcpba  coli  and  Entamceba 
histolytica,  as  described  by  Schaudinn.  It  should  be 
remembered,  however,  that  one  has  to  examine  many 
preparations  before  this  can  be  accomplished  and  that 
the  majority  of  air-dried  preparations  are  almost  use- 
less for  the  study  of  minute  cytological  details. 

The  following  chemicals  are  used  in  preparing 
Wright's  stain: 

1.  Methylene   blue    (Grubler's). 

2.  Eosin.  Yellow.  Water  soluble  (Grubler's). 

3.  Methylic  alcohol  (Merck's  reagent). 

Method  of  Preparation. — The  stain  is  prepared 
as  follows:  Add  0.5  gm.  of  sodium  bicarbonate  to 
100  c.c.  of  distilled  water,  dissolve  thoroughly,  and 


52  PARASITIC  AMGEBJE  OF  MAN. 

add  1  gm.  of  methylene  blue  (Grubler)  ;  heat  the 
mixture  for  one  hour  in  an  Arnold  sterilizer  or  other 
steam  sterilizer,  after  the  steam  is  up.  After  heating 
allow  the  solution  to  cool.  Make  a  1  to  1,000 
solution  of  yellow  aqueous  eosin  (Grubler)  and  add 
this,  while  stirring,  to  the  cooled  methylene  blue  solu- 
tion, in  about  the  proportion  of  500  c.c.  of  the  eosin 
solution  to  75  c.c.  of  the  methylene  blue  solution. 
This  should  be  done  in  a  white  dish  of  some  kind,  in 
order  that  the  precipitate  that  forms  as  the  eosin 
solution  is  added  may  be  easily  seen.  The  proportion 
of  eosin  solution  added  to  the  methylene  blue  solution 
will  vary  in  different  batches  of  the  stain,  but  the 
eosin  solution  should  always  be  added  until  a  well- 
marked  precipitate  follows  after  stirring.  A  marked 
metallic  scum  will  appear  upon  the  surface  of  the 
mixture  at  the  time  the  eosin  solution  is  in  sufficient 
quantity  and  this  may  be  used  as  an  indicator,  but 
if  no  precipitate  is  present  when  the  metallic  scum 
appears  the  eosin  solution  should  be  added  until  it 
appears.  The  mixture  should  then  be  allowed  to 
stand  for  fifteen  or  twenty  minutes,  and  then  filtered 
through  one  small  filter-paper,  the  precipitate  saved, 
dried  in  a  hot-air  oven,  removed  from  the  filter-paper, 
and  used  in  making  the  staining  solution.  The 
precipitate,  which  is  a  fine  powder,  greenish  in  color, 
will  keep  well  and  can  be  used  as  stock  material  for 
months. 


TECHNIQUE.  53 

The  staining  solution  is  prepared  as  follows :  Take 
0.3  gm.  of  the  powdered  precipitate  and  add  it  to  100 
c.c.  of  pure  methylic  alcohol  (Merck's  reagent) ,  filter, 
and  to  80  c.c.  of  the  filtrate  add  20  c.c.  of  the 
methylic  alcohol,  or  if  more  than  80  c.c.  be  left  after 
filtration,  enough  to  bring  the  entire  amount  up  to 
100  c.c.  The  staining  solution  is  now  ready  for  use 
and  will  keep  unimpaired  for  weeks. 

To  stain,  add  a  few  drops  of  the  staining  solution 
to  the  preparation  of  feces  without  preliminary  fixa- 
tion, or  previously  fixed,  while  wet,  with  osmic  acid 
vapor  followed  by  alcohol,  and  let  stand  for  five 
minutes;  then  add  enough  distilled  water  to  cause  a 
slight  metallic  scum  to  form  on  the  surface  of  the 
preparation;  let  stand  for  from  10  minutes  to  an  hour 
and  wash  thoroughly  in  running  distilled  water.  If 
stained  too  deeply,  wash  in  alcohol  and  xylol.  If  it  is 
desired  to  preserve  the  specimens,  mount  in  Canada 
balsam.  The  best  results  are  obtained  with  wet-fixed 
preparations,  but  carefully  stained  air-dried  speci- 
mens give  good  results  for  ordinary  work. 

Other  methods  of  staining  the  amoeba?  in  the  feces, 
or  in  the  pus  from  liver  abscesses,  have  been  recom- 
mended, but  none  of  them  will  be  found  as  satis- 
factory as  those  which  have  been  described. 

TECHNIQUE  OF  STAINING  SECTIONS  OF  TISSUE 
CONTAINING  AMCEB^E. — Sections  of  the  infected  in- 
testine or  of  the  walls  of  liver  abscesses  are  most  use- 


54  PARASITIC  AMCEB^  OF  MAN. 

ful  in  demonstrating  the  relation  that  the  amoebae 
bear  to  the  lesions  which  are  present.  Various  stain- 
ing methods  are  used  for  this  purpose,  the  most  useful 
of  which  is  probably  Mallory's  stain,  which  differen- 
tiates the  amoebae  in  tissues  in  a  very  satisfactory 
manner. 

Mallory's  Method. — Small  pieces  of  tissue  are 
hardened  in  various  strengths  of  alcohol,  as  is  usual, 
and  imbedded  in  paraffin.  As  thin  sections  as  pos- 
sible should  be  cut  and  stained  for  from  3  to  5 
minutes  in  a  saturated  aqueous  solution  of  thionin; 
differentiated  in  a  2  per  cent,  aqueous  solution  of 
oxalic  acid  for  l/2  to  1  minute;  washed  in  water; 
dehydrated  in  absolute  alcohol;  cleared  in  xylol  and 
mounted  in  xylol  balsam. 

With  this  method  the  nuclei  of  the  a<moebae  stain  a 
brownish  red,  while  the  nuclei  of  other  cells  stain 
blue. 

Safranin. — A  good  stain  for  demonstrating  the 
amoebae  in  tissue  is  safranin.  The  tissues  may  be 
hardened  in  alcohol  or  in  Flemming's  or  Zenker's 
fluids,  and  then  imbedded  in  paraffin.  The  following 
solution  of  safranin  is  used  for  staining:  Equal  parts 
of  a  saturated  aqueous  solution  of  "  safranin  0  soluble 
in  water "  and  a  saturated  alcoholic  solution  of 
"  safranin  soluble  in  alcohol." 

The  sections  are  stained  in  this  mixture  for  from 
6  to  24  hours,  washed  in  water,  and  differentiated  in 


TECHNIQUE.  55 

absolute  alcohol,  after  which  they  are  cleared  in  xylol 
and  mounted  in  xylol  balsam. 

Eosin  and  Methylene  Blue  Stain. — This  stain 
gives  good  results  although  it  does  not  differentiate 
the  amoebae  from  the  cells  of  the  tissues.  However, 
to  one  accustomed  to  the  examination  of  such  tissues 
there  is  no  difficulty  experienced  in  recognizing  the 
amoebae.  If  this  stain  is  used  the  sections  should  be 
fixed  in  Zenker's  fluid  and  imbedded  in  paraffin. 

The  method  of  staining  is  as  follows:  Paraffin 
sections  are  stained  in  10  per  cent,  aqueous  solution 
of  eosin  for  from  15  to  30  minutes;  washed  in  water 
until  the  excess  of  eosin  is  removed;  and  stained  in 
Unna's  alkaline  methylene  blue  solution  diluted  5 
times  with  water,  for  from  10  to  15  minutes.  The 
Unna  solution  consists  of  the  following: 

Methylene  blue  (Grubler)    1.0  gram. 

Carbonate  of  potass 1.0  gram. 

Distilled  water 100.0  c.c. 

After  remaining  in  this  solution  for  the  required 
time  the  sections  are  washed  in  water  and  differen- 
tiated and  dehydrated  in  95  per  cent,  alcohol.  This 
procedure  should  be  controlled  under  the  microscope 
and  should  be  continued  until  a  pink  color  has  re- 
turned to  the  section  and  the  nuclei  of  the  cells  appear 
a  deep  blue;  absolute  alcohol  is  then  used  to  com- 
plete the  dehydration,  the  sections  are  cleared  in  xylol, 
and  mounted  in  xylol  balsam.  Care  should  be  taken 


56  PARASITIC  AMOSB.E  OF  MAN. 

that  during  the  first  staining  with  eosin  a  deep  red 
color  be  obtained  as  otherwise  it  will  all  be  removed 
by  the  methylene  blue  solution. 

The  Hoematoxylin-Eosin  Stain. — Sections  may 
be  stained  by  this  method  and  give  fairly  good  results. 
They  should  first  be  fixed  in  Zenker's  solution  and 
then  imbedded  in  paraffin.  The  solution  of  hsema- 
toxylin  used  is  that  known  as  Delafield's  and  is  pre- 
pared as  follows : 

Four  grams  of  haematoxylin  crystals  (Grubler's) 
are  dissolved  in  25  c.c.  of  95  per  cent,  alcohol  and  to 
this  is  added  400  c.c.  of  a  saturated  aqueous  solution 
of  ammonia-alum,  the  mixture  then  being  allowed  to 
stand  in  an  unstoppered  bottle  exposed  to  the  light 
and  air  for  four  days.  It  is  then  filtered  and  to  the 
filtrate  is  added  100  c.c.  of  glycerine  and  the  same 
amount  of  95  per  cent,  alcohol.  This  solution  is 
allowed  to  stand  in  the  light  until  it  has  a  deep  purple 
tinge,  when  it  is  filtered  and  stored  in  a  tightly  stop- 
pered bottle.  If  the  purple  tinge  is  lost  the  solution 
is  no  longer  suitable  for  staining. 

In  using  the  stain  for  section  work  the  sections 
are  stained  in  it  for  30  minutes  to  an  hour,  or  more, 
and  are  then  washed  in  several  changes  of  water  or 
in  running  tap  water  for  from  10  to  30  minutes. 
After  they  are  thoroughly  washed,  an  aqueous  solu- 
tion of  eosin  of  y2  per  cent,  strength  is  used  for  a 


TECHNIQUE.  57 

contrast  stain,  after  which  the  sections  are  dehydrated 
in  95  per  cent,  alcohol,  cleared  in  oleum  origani  cretici, 
and  mounted  in  Canada  balsam.  As  good  results  are 
obtained  by  adding  a  few  drops  of  the  haematoxylin 
solution  to  a  small  dish  full  of  water  containing  the 
sections  and  allowing  them  to  stay  in  this  solution 
over  night,  after  which  the  contrast  stain  is  used  as 
described. 

Heidenhain's  Iron-hcematoxyUn  Method. — Sec- 
tions fixed  with  Zenker's  fluid  and  not  over  6  microns 
in  thickness  may  be  stained  by  the  Heidenhain  method 
in  the  same  manner  as  heretofore  described  for  stain- 
ing smears  of  material  containing  amoeba?. 

A  great  deal  of  care  is  required  in  staining  sec- 
tions containing  amoebae  if  the  best  results  are  to  be 
obtained,  and  sometimes  many  preparations  have  to 
be  stained  before  typical  ones  are  secured.  This  is 
true  of  any  method  which  may  be  adopted,  although 
the  writer  believes  that  the  Mallory  method  is  the 
easiest  of  application  and  gives  the  most  uniformly 
satisfactory  results. 

For  the  diagnosis  of  amoebae  in  the  stools  stain- 
ing methods  should  never  be  resorted  to,  as  the 
examination  of  fresh  specimens  is  altogether  superior 
and  safer.  The  stained  preparations  are  only  used 
in  the  study  of  the  morphological  character  and  the 
methods  of  reproduction  of  these  organisms. 


V. 

THE  CULTIVATION  OF  PARASITIC  AMCEB^E. 

MANY  authorities  have  claimed  to  have  cultivated 
the  parasitic  amoebae  of  man,  while  others  have  in- 
sisted that  such  cultivation  has  not  been  accomplished. 
Anyone  studying  the  literature  is  struck  with  the 
discordant  results  obtained  by  different  observers, 
and  I  believe  that  it  is  now  generally  accepted  by 
zoologists,  who  have  thoroughly  studied  these  organ- 
isms, that  none  of  the  parasitic  amoeba?  of  man  have 
been  cultivated.  While  it  is  not  at  all  difficult  to 
cultivate  free-living  forms,  it  is  very  doubtful,  to  say 
the  least,  if  any  of  the  amoebse  which  have  become 
parasitic  can  be  cultivated  by  the  methods  at  present 
in  vogue. 

It  is  admitted  by  all  that  amoebae  have  been 
cultivated  from  the  feces  of  patients  suffering 
from  dysentery,  but  it  cannot  be  admitted  that 
these  amoebae  are  parasitic  forms;  in  reality  they 
are  free-living  amoeba?  which  have  reached  the 
intestine  in  food  or  drink,  and  have  simply  passed 
through  it  in  an  encysted  condition.  If  cultures  be 
made  from  the  feces  containing  these  free- 
living  species  they  will  develop  if  suitable  bacteria 

58 


CULTIVATION  OF  PARASITIC  AMOEBAE.       59 

be  present  and  it  is  this  fact  which  has  led  to  the 
mistaken  idea  that  the  parasitic  amoebae  are  easily 
cultivated. 

Regarding  this  subject  Braun  and  Liihe,  in  their 
latest  work  upon  parasitology,  published  in  1910,  say : 
'  It  must  be  borne  in  mind,  however,  that  not  all 
amoebge  can  be  cultivated  upon  solid  media,  and  that 
the  varieties  which  are  parasitic  in  mammals  have 
never  been  successfully  grown  outside  of  the  tissues 
of  their  host."  This  statement  is  concurred  in  by 
the  writer,  for  I  have  never  been  able  to  obtain  any 
amoebae  upon  cultural  material  which  resembled  those 
parasitic  in  man,  nor  have  I  ever  seen  amoebae  cul- 
tivated by  others  which  agreed  in  either  their  mor- 
phology or  their  methods  of  reproduction  with  the 
parasitic  species  occurring  in  the  human  host.  All 
cultivated  amoebae  that  I  have  observed  possess  a  con- 
tractile vacuole  and  otherwise  resemble  in  their  mor- 
phology the  common  free-living  species.  The  success- 
ful production  of  dysenteric  lesions  in  animals  by  the 
use  of  cultures  of  amoebae  obtained  from  dysenteric 
feces  proves  nothing,  as  the  material  could  easily  be 
contaminated  by  the  minute  spores  of  Entamceba 
histolytica,  or  encysted  forms  of  other  pathogenic 
species,  or  by  bacteria  capable  of  causing  dysentery. 

In  regions  where  free-living  amoebae  are  common, 
as  in  the  tropics,  it  is  not  at  all  difficult  to  cultivate 


60  PARASITIC  AMCEBJS  OF  MAN. 

amoeba?  from  the  feces  of  man  either  in  health  or 
disease,  but  in  temperate  regions,  where  free-living 
forms  are  comparatively  rare,  it  is  but  seldom  that 
one  is  successful  in  cultivating  such  organisms  from 
the  feces.  Numerous  attempts  have  been  made  to 
cultivate  amoebae  at  this  laboratory  from  patients 
whose  stools  contained  Entamoeba  histolytica  and  En- 
tamoeba tetragena,  as  well  as  Entamoeba  coli,  and  in 
not  a  single  instance  have  cultures  been  obtained 
of  any  amoebae,  in  spite  of  the  fact  that  the  same 
methods  were  used  that  had  proven  successful  in  the 
Philippine  Islands,  and  that  the  same  observers  had 
secured  amoeba  cultures  in  those  Islands  without 
trouble.  However,  it  is  not  difficult  to  explain  these 
results.  In  the  Philippines  almost  all  drinking  water, 
as  well  as  salad  vegetables,  are  infested  with  free- 
living  amoebae,  and  it  naturally  follows  that  such  forms 
are  continually  passing  through  the  intestinal  canal 
of  man,  and  may  be  obtained  upon  suitable  culture 
media  from  the  infected  feces ;  in  Washington,  on  the 
other  hand,  free-living  species  of  amoebae  are  com- 
paratively rare,  and  it  follows  that  attempts  to  culti- 
vate amoebae  from  the  feces  generally  result  in  failure, 
as  the  parasitic  species  do  not  develop  upon  the  cul- 
ture media  at  present  in  use.  To  my  mind  the  failure 
to  cultivate  amoebae  from  dysentery  patients  in  certain 
regions,  although  the  same  methods  which  are  sue- 


CULTIVATION  OF  PARASITIC  AMGEBJE.       61 

cessful  in  other  regions  are  employed,  is  sufficient 
proof  that  the  parasitic  amoebae  of  man  have  not  been 
cultivated.  In  no  other  way  can  we  explain  the  suc- 
cessful results  attending  the  cultivation  of  amoebae  in 
the  tropics  and  the  unsuccessful  results  in  temperate 
regions  even  though  material  be  used  from  patients 
heavily  infected  with  Entamoeba  histolytica,  or  En- 
tamoeba  coli. 

A  review  of  the  literature  on  the  subject  and  of 
the  methods  which  have  been  used  in  cultivating 
amoebaB  is  here  given.  I  do  not  believe  that  any  of  the 
methods  recommended  for  cultivation  have  been  suc- 
cessful so  far  as  the  parasitic  species  of  man  are  con- 
cerned, but  modifications  of  them  may  prove  of 
service  in  further  research  work  upon  these  para- 
sites, while  the  study  of  cultures  of  free-living  species 
is  of  service  in  comparative  morphology. 

Cunningham  in  1879,  and  Grassi  in  1882,  claimed 
to  have  been  able  to  cultivate  dysentery  amoebae  and 
stated  that  such  cultures  produced  dysentery  in  cats. 

In  1890,  Kartulis  attempted  to  cultivate  amoeba? 
from  the  intestinal  discharges  of  cases  of  dysentery 
occurring  in  Egypt  and  stated  that  he  obtained  pure 
cultures  in  sterile  straw  infusion  and  in  feces  diluted 
with  alkaline  bouillon.  Both  his  observations  and 
those  of  Cunningham  and  Grassi  are  now  discredited 
so  far  as  the  cultivation  of  the  parasitic  species  of  man 
is  concerned. 


62  PARASITIC  AMOEBJS  OF  MAN. 

In  1897,  Frosch  demonstrated  that  it  was  pos- 
sible to  grow  free-living  amoebae  upon  culture  media, 
provided  bacteria  were  present  at  the  same  time,  and 
that  unless  such  bacteria  were  present  it  was  impos- 
sible to  carry  the  organisms  from  one  culture  to 
another.  In  the  same  year  Casagrandi  and  Barba- 
gallo  confirmed  these  observations,  but  considered 
that  the  association  of  the  amoebae  and  bacteria  was 
simply  an  accidental  one.  Tsujitani,  in  1898,  cul- 
tivated amoebae  in  association  with  cholera  vibrios,  and 
by  heating  the  cultures  at  60  degrees  C.  he  killed 
the  vibrios,  thus  obtaining  a  pure  culture  of  the 
amoebae.  He  found,  however,  that  while  the  encysted 
amoebae  upon  the  cultures  were  capable  of  motility 
and  growth,  multiplication  did  not  occur  when  they 
were  transferred  to  sterile  culture  media.  His  ob- 
servations, together  with  those  of  Zaubitzer,  Mouton, 
and  Schardinger,  definitely  proved  that  it  is  impossible 
to  transplant  pure  cultures  of  amoebae  free  from  all 
bacteria. 

The  most  extensive  work  done  upon  the  cultiva- 
tion of  the  parasitic  amoebae  of  man  is  that  of  Mus- 
grave  and  Clegg.  In  1904,  these  investigators  pub- 
lished a  monograph  upon  this  subject  in  which  a 
complete  review  was  given  of  the  literature,  together 
with  their  methods  and  results.  They  claim  to  have 
been  able  to  cultivate  dysentery  amoebae  in  symbiosis 


CULTIVATION  OF  PARASITIC  AMCEBJ2.       63 

with  pure  cultures  of  bacteria  and  to  have  produced 
in  monkeys  typical  symptoms  of  the  disease  by  the 
use  of  such  cultures.  While  there  can  be  no  question 
that  they  were  able  to  cultivate  amoebae  from  the  f eces 
of  dysentery  cases  it  is  still  uncertain  just  what  species 
were  obtained  in  this  way,  and  while  lesions  were 
undoubtedly  produced  by  the  mixed  cultures  of 
amoebae  and  bacteria,  the  authors  could  not,  with  their 
methods,  be  sure  of  excluding  the  spores  of  En- 
tamoeba  histolytica,  or  the  encysted  forms  of  other 
amoebae  pathogenic  to  the  animals  used  in  their  experi- 
ments. As  they  did  not  accept  Schaudinn's  classifi- 
cation, they  made  no  differentiation  between  patho- 
genic and  free-living  amoebae,  and  many  of  their 
photomicrographs  of  the  organisms  from  cultures 
prove  that  they  were  dealing  with  ordinary  free-living 
species. 

Walker,  in  1908,  following  and  amplifying  the 
methods  of  Musgrave  and  Clegg,  claims  to  have  suc- 
cessfully cultivated  numerous  amoebae,  including 
parasitic  species,  but  his  description  of  the  life  cycle 
of  the  parasitic  forms  does  not  agree  with  that  of 
Schaudinn,  Doflein,  Ltihe,  Viereck,  and  many  others 
who  have  thoroughly  studied  the  methods  of  repro- 
duction of  amoebae  found  both  in  health  and  disease. 
It  is  more  than  probable  that  the  amoebae  cultivated 
by  Musgrave  and  Clegg,  and  by  Walker,  were  free- 
living  species  occurring  accidentally  in  the  feces. 


64  PARASITIC  AMOEBAE  OF  MAN. 

TECHNIQUE  OF  CULTIVATION. — A  large  number 
of  culture  media  have  been  recommended  for  the  cul- 
tivation of  amoebse.  Kartulis  recommended  a  solution 
of  ordinary  bouillon  and  sterilized  infusions  of  hay; 
Ogata  used  a  2^2  per  cent,  solution  of  grape  sugar 
in  sterilized  water;  Vivaldi  used  sterilized  straw  in- 
fusion, and  Miller  was  able  to  grow  the  organisms 
in  a  1/5  per  cent,  solution  of  milk,  in  water,  in  hay 
infusions,  and  in  dilute  bouillon.  A  number  of  ob- 
servers have  used  solid  media,  such  as  agar,  alkaline 
potatoes,  egg  albumin,  and  5  per  cent,  of  Fucus  cris- 
pus  in  alkaline  albumin.  Among  other  media  which 
have  been  used  may  be  mentioned  Gensen's,  com- 
posed of  barley  sprouts  cooked  in  water,  filtered, 
rendered  alkaline,  and  mixed  with  sugar,  after  which 
it  was  sterilized  by  the  fractional  method;  Zaubitzer's, 
consisting  of  solutions  containing  varying  amounts  of 
somatose  or  agar  containing  from  1  to  2  per  cent, 
somatose;  and  Casagrandi  and  Barbagallo's,  consist- 
ing of  the  sterile  white  of  egg  containing  bicarbonate 
of  soda. 

While  it  is  true  that  free-living  amoebge  may  be 
cultivated  upon  almost  all  of  these  media  the  one 
which  has  given  the  most  satisfactory  results  is  that 
devised  by  Musgrave  and  Clegg,  the  formula  of  which 
is  as  follows : 


CULTIVATION  OF  PARASITIC  AMOEBJC.       65 

Agar   20.0  grams. 

Sodium   chloride    0.3-0.5    gram. 

Extract  of  beef   0.3-0.5  gram. 

Distilled  water   1000.  c.c. 

This  is  prepared  exactly  as  is  ordinary  nutrient 
agar  and  is  made  1  per  cent,  alkaline,  using  phenol- 
phthalein  as  an  indicator.  Upon  this  medium  numer- 
ous species  of  free-living  amoebse  develop,  provided 
suitable  bacteria  are  present  in  symbiosis.  Musgrave 
and  Clegg  have  determined  that  many  amoebae  are 
very  selective  in  regard  to  the  bacteria  with  which  they 
will  develop,  and  they  regard  failures  to  grow  amoebse 
as  largely  due  to  the  absence  of  a  suitable  symbiotic 
bacterium. 

Because  of  this  selective  action  of  the  amoebae  it 
is  possible  to  grow  them  in  pure  culture  with  a  single 
species  of  bacterium  and  these  cultures  are  known  as 
:<  pure  mixed  cultures  of  amoebae."  Of  the  bacteria 
which  have  been  found  most  suitable  for  cultivation 
with  amoebae  may  be  mentioned  Bacillus  coti,  Vibrio 
cholerce,  Bacillus  typliosus,  Staphylococcus  pyogenes 
aureus  and  such  non-pathogenic  organisms  as  Bacillus 
fluorescens  and  Bacillus  rubra. 

It  is  a  significant  fact  that  in  most  of  the  experi- 
ments resulting  in  the  production  of  pathogenic  lesions 
by  the  use  of  cultures  of  amoebae,  the  bacteria  grown  in 
symbiosis  with  the  amoebae  have  been  pathogenic 
species.  For  this  reason  the  results  must  be  regarded 


66  PARASITIC  AMCEB^E  OF  MAN. 

as  doubtful  as  one  can  hardly  draw  conclusions  re- 
garding the  pathogenicity  of  the  amoebae  from  the 
results  of  injection  into  animals  of  these  mixed  cul- 
tures containing  both  amoeba?  and  pathogenic  bacteria. 

Cultures  from  Water. — The  method  recommended 
by  Musgrave  and  Clegg  for  securing  cultures  of 
amoebae  from  water  is  the  following: 

From  100  to  500  c.c.  of  the  water  to  be  examined 
is  collected  in  a  sterile  flask  and  y2  to  1  c.c.  of  1  per 
cent,  alkaline  bouillon  is  added  to  each  100  c.c.  of  the 
sample.  The  flask  is  stoppered  with  cotton,  and 
placed  in  the  dark  for  from  48  to  72  hours,  and  at 
the  end  of  this  time  a  loopful  of  the  water  from  the 
surface  is  examined  for  amoebge.  A  loopful  is  then 
spread  over  the  surface  of  the  medium  used,  prefer- 
ably that  described  above,  which  has  been  poured  into 
a  Petri  dish.  The  plate  should  be  examined  at  the  end 
of  24  hours,  and  in  many  instances  amoebae  will  be 
found  at  that  time,  but  sometimes  several  days  elapse 
before  the  cultures  develop.  It  is  not  necessary  to 
use  a  symbiotic  bacterium,  as  the  amoebae  will  find 
suitable  bacteria  in  the  water. 

Cultures  from  Feces. — For  the  cultivation  of 
amoebae  from  feces  the  following  method  is  recom- 
mended by  Musgrave  and  Clegg: 

The  surface  of  the  special  culture  medium,  con- 
tained in  Petri  dishes,  is  smeared  with  a  culture  of 


CULTIVATION  OF  PARASITIC  AMCEB^E.       67 

Bacillus  coli  or  some  other  bacterium  with  which 
amoebae  grow  well,  and  the  feces  is  then  smeared  in 
concentric  circles  upon  the  surface  of  the  medium  or  in 
streaks  across  it.  The  plates  are  then  kept  at  room 
temperature  and  examined  upon  successive  days  for 
at  least  a  week.  The  amoebae  can  be  easily  seen  upon 
the  plates,  as  they  wander  from  the  material  in  the 
spread  out  into  the  surrounding  medium  and  the 
course  that  they  take  can  often  be  followed  by  the 
development  of  colonies  of  the  bacteria,  which  they 
carry  with  them. 

Both  Musgrave  and  Walker  state  that  the  de- 
velopment of  the  parasitic  species  occurs  more 
readily  at  room  temperature  than  in  the  incubator 
at  37°  C.  As  such  species  are  normally  subjected 
to  the  temperature  of  the  body,  it  is  difficult  to  under- 
stand why  they  should  grow  best  under  artificial  con- 
ditions at  a  lower  temperature.  This  is  certainly  not 
true  of  any  other  protozoon  and  suggests  that  all 
their  cultivated  amoebae  were  really  free-living  forms. 
In  every  instance  where  cultural  forms  of  the  protozoa 
have  been  obtained,  in  which  growth  occurs  at  a  lower 
temperature  than  that  of  the  host,  such  parasites 
have  depended  for  transmission  upon  some  insect  hav- 
ing a  low  body  temperature.  There  is  no  evidence 
that  the  parasitic  amoebae  undergo  ^ny  development 
in,  or  are  transmitted  by,  an  insect,  and  I  consider  that 


68  PARASITIC  AMOEBA  OF  MAN. 

the  failure  of  so-called  parasitic  amoebse  to  develop 
upon  culture  media  at  the  body  temperature  is  addi- 
tional proof  of  the  free-living  nature  of  the  amoebse 
which  have  been  cultivated.  However,  even  free- 
living  amoebae  may  be  cultivated  at  37°  C.  without 
much  difficulty. 

A  simple  and  easy  method  of  securing  cultures  of 
amoeba?  from  feces  consists  in  mixing  with  the  ma- 
terial to  be  examined  an  emulsion  of  the  bacterium 
to  be  used  in  symbiosis  and  streaking  this  mixture 
across  the  surface  of  the  culture  medium  contained 
in  Petri  dishes. 

To  secure  a  culture  of  a  single  amoeba  in  symbiosis 
with  bacteria  the  culture  plates  are  carefully  ex- 
amined and  a  single  amoeba  is  located.  In  order  to 
remove  this  organism  from  the  plate,  Musgrave's 
method  may  be  employed,  which  consists  in  lowering 
a  low  power  objective  until  it  just  touches  the  medium 
where  the  amoeba  lies,  and  then  quickly  raising  it, 
thus  picking  up  the  amoeba,  which  can  then  be  trans- 
ferred to  a  new  plate ;  or  a  single  organism  is  located 
and  with  a  fine  glass  capillary  pipette  is  removed 
from  the  surface  of  the  plate,  and  placed  upon  new 
media.  Walker  marks  the  situation  of  the  single 
amoeba  with  a  wax  pencil  on  the  bottom  of  the  Petri 
dish,  using  a  V-shape  mark,  the  apex  of  which  just 
encloses  the  amoeba,  the  arms  of  the  V  extending 


CULTIVATION  OF  PARASITIC  AMOEBAE.       69 

away  from  the  streak  of  bacterial  growth;  the  plate 
is  then  turned  right  side  up,  the  cover  removed,  and 
with  carbolated  vaseline  the  V-shape  mark  is  traced 
on  the  surface  of  the  medium.  In  this  manner  the 
single  amoeba  is  isolated  and  can  be  transferred,  after 
multiplication,  to  a  new  plate.  It  is  well  to  cover  the 
surface  of  the  medium  outside  the  arms  of  the  V  with 
a  little  additional  vaseline. 

The  Cover-glass  Method  of  Cultivation. — This 
very  ingenious  method  of  cultivating  amoebse  was  de- 
vised by  Walker  and  is  thus  described  by  him: 

"  It  is  based  on  the  principle  of  Koch's  original 
plate  culture  for  bacteria,  is  indeed  a  miniature  Koch's 
plate  culture  made  on  a  cover-glass  and  inverted  over 
a  concave  slide.  For  this  reason  I  have  called  such 
culture  a  *  hanging  plate  culture.'  This  culture  is 
prepared  as  follows:  A  thin  seven-eighths-inch  cover 
glass  is  flamed  and  placed  under  a  flamed  watch  glass. 
With  a  sterile  loop  a  large  loopful  of  the  liquefied 
agar  medium  is  transferred  to  the  sterile  cover,  and 
spread  in  a  uniform,  thin  and  circumscribed  layer. 
This  film  of  agar  will  solidify  almost  instantly  and 
it  is  at  once  inoculated  with  the  amoeba  culture.  The 
cover-glass  culture  is  then  inverted  over  the  concave 
centre  of  a  hollow  slide,  which  has  been  previously 
flamed,  ringed  with  vaseline,  and  kept  in  an  inverted 
position  near  at  hand.  This  transfer  of  the  cover- 


70  PARASITIC  AMCEB.E  OF  MAN. 

glass  culture  to  the  slide  can  be  accomplished  either 
by  picking  up  the  cover  with  forceps  and  inverting 
it  over  the  concave  centre  of  the  slide,  or  by  pressing 
the  inverted  slide  upon  the  cover  glass,  which  adheres 
to  the  vaseline  ring  around  the  concavity  of  the  slide. 
The  cover  is  then  pressed  down  around  the  edges 
until  the  vaseline  completely  seals  the  culture  so  as 
to  prevent  evaporation  of  moisture.  Ordinarily  the 
moisture  of  condensation  from  the  solidifying  agar 
and  that  transferred  in  inoculating  the  culture  is  suffi- 
cient for  growth,  but  sterile  distilled  water  or  any 
media  or  reagents  can  be  added  with  a  sterile  loop, 
either  at  the  time  of  making  the  culture  or  later  by 
raising  the  cover.  On  these  cover-glass  cultures 
amoebae  multiply  as  freely  as  on  Petri  plate  cultures. 
The  film  of  agar  medium  is  thin  enough  to  permit  the 
use  of  a  one-twelfth-inch  oil  immersion  objective,  and 
is  so  nearly  transparent  that  it  does  not  seriously  ob- 
struct the  passage  of  light." 

According  to  Walker  the  following  are  the  essen- 
tial factors  in  the  cultivation  of  amoebae  on  artificial 
media:  (1)  the  consistence  should  be  solid;  (2)  re- 
action should  be  neutral  or  preferably  slightly  alka- 
line; (3)  bacteria  should  be  present  on  which  the 
amoebae  can  feed;  (4)  media  should  be  moist  and 
oxygen  should  be  present;  (5)  a  temperature  of  20 
to  25°  C.  should  be  maintained. 


CULTIVATION  OF  PARASITIC  AMCEBJS.       71 

For  a  full  discussion  of  the  methods  used  in  the 
past  and  at  present  for  the  cultivation  of  amoebae  the 
reader  is  referred  to  the  work  of  Musgrave  and  Clegg 
and  to  that  of  Walker. 

I  desire  here  to  protest  against  the  growing 
tendency  of  drawing  conclusions  regarding  the  mor- 
phology and  the  life  cycle  of  the  parasitic  amoebae  as 
observed  in  man  from  organisms  growing  upon  arti- 
ficial culture  media.  Even  if  it  were  definitely  proven 
that  any  of  the  parasitic  amoebae  have  been  cultivated, 
deductions  based  upon  the  appearance  of  the  amoebae 
in  such  cultures  would  probably  be  erroneous,  as  it  is 
well  known  that  the  cultural  forms  of  protozoa  so 
far  described  differ  markedly  in  their  morphology 
and  life  history  from  the  forms  observed  in  the  human 
host.  When  we  add  to  this  fact  the  doubt  which 
exists  as  to  the  cultivation  of  the  parasitic  species 
it  is  obvious  that  any  attempt  to  describe  or  to  liken 
the  morphology  of  cultural  forms  to  the  parasites  ob- 
served in  man  must  result  in  failure  and  confusion. 
The  entire  subject  of  the  cultivation  of  the  parasitic 
amoebae  is  in  a  chaotic  condition  and  much  more  work 
will  have  to  be  done  before  it  can  be  accepted  that 
any  of  the  parasitic  species  of  man  have  been  cul- 
tivated. 

Major  Whitmore  of  the  U.  S.  Army  Medical 
Corps,  who  has  recently  worked  with  Hartmann. 


72  PARASITIC  AMGEBJE  OF  MAN. 

states  in  a  letter  to  the  author,  that  all  of  the  cultures 
of  amoeba?  he  obtained  in  Manila  from  dysenteric 
patients  both  from  the  feces  and  from  liver  abscess 
pus,  as  well  as  cultures  from  water,  were  found,  in 
Hartmann's  laboratory,  to  be  free-living  amoebge  and 
of  no  etiological  significance  in  dysentery.  This  is  in 
accordance  with  what  I  have  always  believed  and 
stated,  i.e.,  that  the  parasitic  amoebae  of  man  have  not 
been  cultivated. 


VI. 

THE  AMGEBJE  OF  THE  INTESTINAL  TRACT. 

THE  following  amoebae  have  been  described  as 
parasitic  within  the  intestinal  tract  of  man: 

Genus,  Entamoeba. 

Entamoeba   coli,   Loesch,   1875,   emend 

Schaudinn,  1903. 

Entamoeba  Mstolytica,  Schaudinn,  1903. 
Entamceba  tetragena,  Viereck,  1907- 
Entamoeba  minuta,  Elmassian,  1909. 
Entamceba  nipponica,  Koidzumi,  1909. 
Entamoeba  tropicalis,  Lesage,  1908. 
Entamoeba  phagocytoides,  Gauducheau, 

1908. 
Entamoeba  undulans,  Castellani,  1905. 

Genus,  Paramoeba. 

Par  amoeba  hominis,  Craig,  1906. 

ENTAM(EBA  COLL     Loesch,  1875.     Emend  Schaudinn,  1903. 

Before  the  publication  of  Schaudinn's  researches 
observers  had  noticed  the  occurrence  of  amcebse  in  the 
intestinal  discharges  of  healthy  individuals  and  those 
suffering  from  diseases  other  than  dysentery,  and 
this  gave  rise  to  the  opinion  that  these  organisms  were 

73 


74  PARASITIC  AMCEB^E  OF  MAN. 

harmless  and  only  of  accidental  occurrence  in  patients 
suffering  from  dysentery.  However,  as  evidence  of 
the  pathogenic  action  of  amoeba?  accumulated  it  was 
found  that  such  an  opinion  was  untenable.  In  order 
to  explain  the  pathogenic  action  of  these  organisms 
the  theory  regarding  the  effect  of  environment  arose, 
together  with  the  belief  that  more  than  one  species 
of  amoebae  occurred  in  the  human  intestine. 

OCCURRENCE  IN  HEALTHY  INDIVIDUALS. — It  is 
surprising  how  little  work  has  been  done  regarding 
the  occurrence  of  amoebae  in  the  healthy  human  in- 
testine. Although  several  years  have  elapsed  since 
Schaudinn's  paper  was  published,  only  a  very  few  ob- 
servers have  undertaken  the  determination  of  how 
large  a  percentage  of  healthy  individuals  show  amoebae 
in  the  feces,  and  even  those  who  have  combated 
Schaudinn's  classification  have  not  taken  the  trouble 
to  examine  a  large  number  of  individuals  in  health, 
in  order  to  prove  or  disprove  his  results. 

Historical  Summary. — Grassi,  in  1888,  was  prob- 
ably the  first  investigator  to  demonstrate  the  presence 
of  amoebae  in  the  feces  of  healthy  individuals,  although 
Cunningham  in  1881  stated  that  he  found  amoebae  in 
the  stools  in  both  healthy  and  diseased  individuals, 
but  his  description  indicates  that  he  mistook  stages 
in  the  life  cycle  of  the  flagellates  for  amoebae.  Shu- 
berg  demonstrated  amoebae  in  the  feces  of  10  out  of 


AMGEB.E  OF  THE  INTESTINAL  TRACT.       75 

20  healthy  individuals,  or  50  per  cent.,  while  Gassard 
found  20  per  cent,  of  infections  in  healthy  individuals 
in  examining  20  cases.  Strong  and  Musgrave  found 
amoebae  in  only  4  per  cent,  of  healthy  individuals,  but 
felt  justified  in  believing  that  these  amoebae  were  not 
pathogenic  because  of  the  negative  results  of  animal 
experiments.  Dock  examined  200  healthy  people  and 
only  found  amoebae  in  2  cases.  He  concludes  his  con- 
tribution to  this  subject  as  follows :  "Even  if  a  certain 
parasite  occurs  in  every  case  in  one  locality,  it  would 
not  follow  that  the  same  parasite  would  also  be  found 
as  widespread  elsewhere."  Schaudinn,  in  1903,  pub- 
lished the  results  obtained  by  him  in  the  examination 
of  the  feces  of  healthy  individuals.  He  found  that 
in  West  Prussia  50  per  cent,  of  healthy  men  among 
the  farming  population  showed  amoebae  in  their  feces, 
while  in  Berlin  only  20  per  cent,  were  found  infected; 
along  the  shores  of  the  Adriatic  Sea  he  found  that 
in  385  examinations  of  as  many  individuals  in  per- 
fect health,  no  less  than  256  or  66  per  cent,  showed 
the  presence  of  amoebae.  His  observations,  as  well 
as  those  of  others,  demonstrate  that  locality  and 
occupation  have  much  to  do  with  the  number  of  in- 
dividuals infected.  In  all  the  cases  of  healthy  in- 
dividuals examined  by  him  the  amoebae  found  was 
Ent amoeba  coli. 

In    1905,    I    published    the    results    obtained    in 


76  PARASITIC  AMOEBA  OF  MAN. 

the  examination  of  healthy  American  soldiers  sta- 
tioned at  San  Francisco,  California.  The  examina- 
tions were  made  largely  among  the  members  of  the 
Hospital  Corps  of  the  U.  S.  Army,  recruited  from 
almost  every  portion  of  the  United  States,  and  who 
were  on  duty  at  the  General  Hospital,  and  under 
constant  observation.  Over  200  men  were  examined 
and  it  was  found  that,  after  the  administration  of 
magnesia  sulphate  in  ounce  doses,  the  f  eces  of  65  per 
cent,  showed  the  presence  of  Entamceba  coll.,  although 
in  many  instances  repeated  examinations  were  neces- 
sary in  order  to  demonstrate  the  parasite.  It  is  the 
writer's  belief  that  the  small  percentage  of  positive 
results  in  the  demonstration  of  this  parasite,  reported 
by  some  observers,  are  due  to  the  examination  of  only 
one  or  two  preparations.  At  least  eight  specimens 
should  be  examined  before  one  can  be  sure  that  En- 
tamoeba coli  is  absent. 

Believing  that  geographic  distribution  might  have 
something  to  do  with  the  proportion  of  infected  in- 
dividuals, the  locality  from  which  each  individual 
came  was  ascertained,  but  there  was  apparently  no 
great  variation  due  to  this  cause.  However,  this 
negative  result  cannot  be  considered  as  conclusive  and 
the  question  can  only  be  settled  when  we  have  data 
concerning  the  examination  of  a  large  number  of  in- 
dividuals in  different  localities.  All  the  men  examined 


AMOEBA  OF  THE  INTESTINAL  TRACT.       77 

were  in  robust  health,  had  never  suffered  from  diar- 
rhoea or  dysentery,  and  presented  no  symptoms  of 
either  of  these  diseases.  In  some  the  amoeba?  occurred 
in  great  numbers  while  in  others  a  long  search  was 
required  before  they  were  demonstrated. 

My  results  were  confirmed,  in  1906,  by  Captain 
Vedder  of  the  Army  Medical  Corps,  who  examined 
the  feces  of  50  healthy  American  soldiers  and  50 
Filipino  Scouts,  the  examinations  being  made  in 
the  Philippine  Islands;  of  the  American  soldiers 
50  per  cent,  showed  Entamceba  coli  in  their  feces, 
while  75  per  cent,  of  the  Filipino  Scouts  were  found 
to  be  infected  with  this  parasite.  Of  the  subsequent 
history  of  the  cases  examined  by  him,  Vedder  says: 
"  All  the  men  have  been  under  observation  for  a  period 
of  nine  months,  and  none  of  them  has  developed 
dysentery." 

Major  Ashburn  and  I,  while  serving  in  Manila 
upon  the  "  Army  Board  for  the  Study  of  Tropical 
Diseases,"  examined  107  healthy  American  soldiers, 
all  of  whom  were  members  of  the  Hospital  Corps  of 
the  Army  and  all  on  active  duty  at  the  U.  S.  Army 
Division  Hospital,  Manila,  P.  I.  Of  the  107  men,  76 
of  71+  per  cent,  were  found  to  be  infected  with  En- 
t amoeba  coli,  while  two  showed  the  pathogenic  En- 
tamceba  histolytica  in  their  stools.  None  of  these 
men,  with  the  exception  of  the  two  showing  the  patho- 


78  PARASITIC  AMOEBAE  OF  MAN. 

genie  amoeba,  had  diarrhoea  or  dysentery  at  the  time 
of  examination,  and  all  denied  ever  having  suffered 
from  dysentery  symptoms  since  residing  in  the 
Philippines.  Of  seventy-two  men  showing  Entamceba 
coli  in  their  feces,  one  had  resided  in  the  Philippine 
Islands  for  eight  years;  four,  seven  years;  one,  six 
and  a  half  years;  three,  six  years;  four,  five  and  a 
half  years ;  one,  five  and  one  quarter  years ;  two,  five 
years ;  four,  four  years ;  three,  three  years ;  two,  two 
and  a  half  years ;  ten,  two  years ;  one,  one  year  and 
ten  months;  two,  one  year  and  nine  months;  nine, 
one  and  a  half  years;  thirteen,  one  year;  and  the 
remainder,  or  seventeen,  less  than  one  year. 

The  two  men  showing  Entamceba  Mstolytica  in 
their  stools  were  apparently  in  good  health,  but  in- 
quiry elicited  the  information  that  both  were  suffering 
from  dysenteric  symptoms  at  the  time  of  examination, 
and  both  were  later  returned  to  the  United  States 
suffering  from  chronic  amoebic  dysentery.  At  the 
time  that  we  examined  the  feces  of  these  men  we 
knew  nothing  of  the  occurrence  of  the  dysenteric 
symptoms,  and  our  diagnosis  was  based  entirely  upon 
the  morphology  of  the  amoebae  observed  in  their  feces. 
It  will  thus  be  seen,  that  contrary  to  the  opinion  of 
certain  investigators,  it  is  possible  to  differentiate 
Entamceba  coli  from  Entamceba  Mstolytica  as  they 
occur  in  the  feces,  and  therefore,  that  such  diff eren- 


AM(EB.E  OF  THE  INTESTINAL  TRACT.       79 

tiation  becomes  of  very  great  importance  in  the  diag- 
nosis of  diarrhoeal  conditions  of  the  intestine. 

In  order  to  determine  how  long  infection  with 
Entamceba  coli  might  exist  we  made  the  following 
examinations : 

A.  Upon  November  20,  1906,  thirteen  men  were 
re-examined    who    had    been    first    examined    upon 
March  17,  1906,  eight  months  having  elapsed  since 
the  first  examination.    Of  these  thirteen  men,  eleven 
showed  Entamosba  coli  in  their  feces  March  17,  and 
nine  or  81.8  per  cent,  still  showed  them  upon  Novem- 
ber 20.    During  this  time  not  one  of  these  men  had 
suffered  from  diarrhoea,  and  all  had  been  on  duty 
continuously  at  the  hospital. 

B.  Upon  November  20,  1906,  seven  men  were 
re-examined  who  were  first  examined  upon  May  2, 
1906,  six  months  and  twenty-two  days  having  elapsed 
since  the  first  examination.    Of  these  seven  men,  five 
were  positive  for  Entamceba  coli  upon  May  2,  and 
five  were  still  positive  upon  November  20,  and  none 
of  these  men  had  suffered  from  diarrhoea  or  dysentery 
during  this  time,  and  were  continuously  under  ob- 
servation. 

C.  Upon  November  20,   1906,  eight  men  were 
re-examined  who  were  first  examined  upon  July  10, 
1906,  four  months  and  thirteen  days  having  elapsed 
since  the  first  examination.    Of  these  eight  men,  five 


80  PARASITIC  AMGEBJE  OF  MAN. 

were  positive  for  Entamoeba  coli  upon  July  10,  and 
two  were  still  positive  upon  November  20.  Neither 
of  these  men  had  developed  symptoms  of  diarrhoea 
or  dysentery  during  this  time. 

As  the  result  of  our  work  we  concluded  that  a 
large  proportion  of  healthy  white  men  serving  in  the 
Philippines  harbored  Entamoeba  coli,  which  infection, 
so  far  as  we  were  able  to  observe,  does  not  result  in 
diarrhoea  or  dysentery,  some  of  the  cases  being  ob- 
served for  9  months  without  any  symptoms  develop- 
ing. 

Summing  up  the  observations  of  the  writer  in 
San  Francisco,  in  1905,  and  those  made  in  conjunc- 
tion with  Ashburn  in  Manila,  we  have  the  record  of 
307  examinations  of  as  many  healthy  American 
soldiers,  of  whom  176  or  58+  per  cent,  showed  the 
presence  of  Entamoeba  coli  in  their  feces.  Some  of 
these  cases  have  been  followed  for  a  period  of  three 
years  and  in  none  of  them  have  symptoms  of  dysen- 
tery occurred.  I  have  observed  instances  in  which 
Entamoeba  coli  has  been  constantly  present  in  the 
feces  for  periods  of  from  4  to  6  years,  and  not  the 
slightest  symptoms  of  dysentery  have  developed. 

In  attempting  to  disprove  the  existence  of  En- 
tamosba  coli  Musgrave  has  instanced  cases  in  which 
amoeba?  were  found  in  the  feces  during  apparent 
health,  but  in  which  dysentery  followed  at  periods 


AMCEBJE  OF  THE  INTESTINAL  TRACT.       81 

varying  from  2  to  6  months,  and  other  cases  in  which 
death  had  occurred  from  other  diseases  and  at  autopsy 
the  intestine  was  found  to  present  the  usual  lesions 
of  amoebic  dysentery,  although  the  patient  had  never 
complained  of  symptoms  of  that  disease.  As  regards 
the  first  class  of  cases  it  may  be  said  they  are  of  no 
scientific  value  as  proof  that  the  amoebae  present  in 
the  beginning  were  pathogenic,  for  the  patients  were 
continually  exposed  to  infection  with  Entamceba 
liistolytica,  in  the  intensely  infected  Philippine 
Islands;  as  regards  the  latter  class  of  cases  it  is 
notorious  that  the  Filipino  frequently  suffers  from 
diarrhoea,  although  he  generally  does  not  consider  this 
symptom  of  enough  importance  to  mention  when 
questioned,  and  most  of  the  instances  reported  have 
been  in  Filipinos.  After  a  long  and  extensive  ex- 
perience, acquired  at  the  autopsy  table,  it  is  my 
opinion  that  it  is  impossible  for  extensive  ulcerative 
lesions  to  occur  in  the  intestine  without  producing 
symptoms  of  dysentery  which  would  attract  the  atten- 
tion of  the  patient.  In  cases  where  the  lesions  are 
localized  in  a  small  area  the  patient  might  pass  a 
small  amount  of  mucus  or  blood  without  knowing  it, 
but  such  cases  possess  no  value  as  proof  of  the  non- 
existence  of  a  harmless  amoeba,  as  both  harmless  and 
pathogenic  species  might  occur  in  such  individuals. 
Until  it  can  be  demonstrated  that  the  lesions  are  the 


82  PARASITIC  AMGEB.E  OF  MAN. 

result  of  infection  with  Entamceba  coli,  we  cannot 
accept  them  as  proof  of  the  pathogenic  character  of 
this  species. 

Since  the  observations  of  Schaudinn  and  myself, 
numerous  investigations  have  confirmed  the  occur- 
rence of  amoeba?,  answering  to  the  description  of 
Entamceba  coli,  in  a  large  proportion  of  healthy 
individuals.  I  believe  that  a  considerable  propor- 
tion of  healthy  individuals  in  almost  any  locality 
will  be  found  to  harbor  Entamoeba  coli  if  the 
examination  is  carefully  made.  Schaudinn  found 
this  species  in  50  per  cent,  of  healthy  individuals 
examined  in  West  Prussia;  in  20  per  cent,  examined 
in  Berlin,  and  in  66  per  cent,  of  those  examined  along 
the  shores  of  the  Adriatic;  Craig  in  65  per  cent,  of 
healthy  American  soldiers  at  San  Francisco;  Vedder 
in  50  per  cent,  of  healthy  American  soldiers  and  in 
72  per  cent,  of  Filipino  Scouts  in  Mindanao,  P.  I.; 
and  Ashburn  and  Craig  in  71  per  cent,  of  healthy 
American  soldiers  in  Manila,  P.  I.  Our  observations 
have  since  been  confirmed  by  Major  Whitmore,  who 
found  this  parasite  present  in  the  Philippines,  as 
well  as  by  Hartmann.  The  percentage  varies  in  dif- 
ferent localities,  but  it  is  evident  that  Entamceba  coli 
is  a  common  parasite  of  man  and  that  it  does 
not  produce  the  symptoms  or  lesions  of  amoebic 
dysentery. 


AMCEB.E  OF  THE  INTESTINAL  TRACT.       83 
OCCURRENCE  or  ENTAMCEBA  COLI  IN  DISEASES 

OTHER    THAN    DYSENTERY. As    Would    be    expected, 

this  species  of  amoebse  not  only  occurs  in  health,  but 
also  in  many  patients  suffering  from  diseases  other 
than  dysentery.  Any  condition  which  leads  to  the 
production  of  acute  or  chronic  diarrhoea,  such  as 
acute  or  chronic  enteritis,  typhoid  fever,  or  cholera, 
is  often  accompanied  by  the  appearance  of  this  para- 
site in  the  feces.  Even  in  conditions  in  which  lesions 
are  not  present  in  the  intestine  this  parasite  is  fre- 
quently observed  in  the  feces  following  the  adminis- 
tration of  a  cathartic.  Cunningham  found  amoebae 
in  the  intestinal  contents  of  cholera  cases;  Celli  and 
Fiocca  in  infants  suffering  from  intestinal  inflamma- 
tion; Berndt  in  cases  of  typhoid  fever;  Normand  in 
chronic  colitis;  Massiutin  in  the  feces  of  5  cases  suf- 
fering with  intestinal  catarrh,  typhoid  fever,  and 
diarrhoea;  Grassi  in  diarrhoea;  Perroncito  in  chronic 
diarrhoea;  Babes  in  cases  of  hepatitis;  Casagrandi 
and  Barbagallo  in  various  diseases,  as  well  as  Kruse 
and  Pasquale,  Quincke  and  Roos,  Kartulis,  Shuberg, 
Gros,  and  Ijima. 

In  my  own  observations  I  have  not  confined  my- 
self to  the  examination  of  feces  of  patients  suffer- 
ing from  diarrhoea,  but  have  examined  the  excreta  in 
all  cases,  whatever  the  diagnosis.  Over  250  such  cases 
have  been  examined  of  which  49  per  cent,  showed 


84  PARASITIC  AMGEB^E  OF  MAN. 

the  presence  of  Entamoeba  coli.  It  is  significant  that 
a  smaller  percentage  of  cases  of  disease  show  this 
amoeba  than  of  healthy  individuals,  which  would  ap- 
pear to  indicate  that  the  parasite  finds  a  more  con- 
genial environment  for  its  development  in  the  in- 
testine of  normal  individuals.  In  all  the  cases  in 
which  diarrhoea  was  not  present  a  saline  cathartic  was 
given  before  the  examination,  and  in  the  patients  in 
whom  diarrhoea  was  present  the  occurrence  of  dysen- 
tery was  excluded  by  the  fact  that  they  were  under 
observation  for  weeks  or  months,  during  which  time 
no  dysenteric  symptoms  developed. 

Table  I  gives  the  results  obtained  in  the 
examination  of  30  cases  of  various  diagnosis,  and 
is  representative  of  the  class  of  cases  examined. 

The  occurrence  of  such  a  large  number  of  in- 
stances of  infection  with  Entamoeba  coli  in  patients 
suffering  from  diseases  other  than  dysentery  is  of 
the  greatest  importance,  especially  in  regions  where 
amoebic  dysentery  is  endemic.  The  examination  of 
the  feces  of  patients  coming  from  such  regions  will, 
in  a  considerable  proportion  of  the  cases,  result  in 
the  finding  of  this  parasite,  and  an  inexperienced 
observer  would  probably  regard  it  as  Entamoeba 
Mstolytica,  and  a  diagnosis  of  amoebic  dysentery 
would  be  made.  It  is  my  belief  that  many  prac- 
titioners have  made  this  mistake  and  that  some 


AMCEB.E  OF  THE  INTESTINAL  TRACT.       85 


of  the  marvellous  cures  attributed  to  certain  remedies 
can  be  accounted  for  in  this  way.  I  have  repeatedly 
observed  cases  diagnosed  as  amoebic  dysentery  upon 
the  demonstration  of  Entamaeba  coli  in  the  stools, 

TABLE  I. 
EXAMINATION  OF  PATIENTS  FOR  ENTAM<EBA  COLI.* 


Number. 

Diagnosis. 

Residence. 

Dysentery. 

Diarrhoea. 

In  hospital. 

Bowel 
move- 
ments. 

E.  Coli. 

1 
2 
^ 

Anemia,  secondary 
Abscess  of  leg 
Abscess  of  axilla 

England 
Illinois 

No 
No 
No 

No 
No 
No 

70  days 
5  months 
20  days 

2  daily 
2  daily 
2  daily 

Present 
Present 
Present 

4 
5 

(; 

Diabetes  insipidus 
Fracture 

Georgia 
Alabama 

No 
No 
No 

No 
No 

No 

40  days 
40  days 
2  months 

1  daily 
2  daily 
2  daily 

Present 
Present 
Present 

7 

Gastritis,  chronic 

No 

No 

2  weeks 

1  daily 

Present 

8 
9 
10 
11 
12 
13 
14 

Gastritis,  acute 
Gonorrhoea,  acute 
Gonorrhoea,  acute 
Gonorrhoea,  acute 
Gonorrhoea,  acute 
Gonorrhoea,  chronic 
Gonorrhoea   chronic 

California 
Tennessee 
California 
Jamaica 
Penna. 
Kentucky 

No 
No 
No 
No 

No 
No 
No 

No 
No 
No 
No 
No 
No 
No 

1  week 
1  month 
1  month 
10  days 
20  days 
35  days 
1  month 

1  daily 
1  daily 
1  daily 
2  daily 
1  daily 
2  daily 
1  daily 

Present 
Present 
Present 
Present 
Present 
Present 
Present 

15 
16 
17 

Gonorrhoea,  chronic 
Hemiplegia 

Penna. 
California 

No 
No 
No 

No 
No 

No 

1  month 
10  days 
10  days 

1  daily 
1  daily 
1  daily 

Present 
Present 
Present 

18 
19 
20 
°1 

Malarial  Fever 
Malarial  Fever 
Malarial  Fever 

Conn. 
California 
Virginia 

No 
No 
No 
No 

No 
No 
No 
No 

2  weeks 
2  weeks 
2  weeks 
2  weeks 

1  daily 
2  daily 
1  daily 
1  daily 

Present 
Present 
Present 
Present 

22 
23 
24 
25 
26 
27 
28 
29 
30 

Otitis  Media 
Measles 
Measles 
Pemphigus 
Poliomyelitis 
Mitral  Regurgitation 
Stricture 
Sciatica,  chronic 
Varicocele 

Texas 
Texas 
Texas 
Arkansas 
Miss. 
Penna. 
California 
Missouri 
Ohio 

No 
No 
No 
No 
No 
No 
No 
No 
No 

No 
No 
No 
No 
No 
No 
No 
No 
No 

3  months 
40  days 
4  months 
5  months 
4  months 
10  days 
2  weeks 
3  months 
3  months 

1  daily 
1  daily 
2  daily 
1  daily 
2  daily 
2  daily 
1  daily 
1  daily 
2  daily 

Present 
Present 
Present 
Present 
Present 
Present 
Present 
Present 
Present 

*  This  table  includes  only  cases  in  which  E.  coli  was  found,  in  order  to  illustrate 
the  variety  of  conditions  in  which  this  parasite  may  be  present. 

after  a  dose  of  salts,  in  which  neither  the  clinical 
history  nor  the  symptoms  corroborated  the  diagnosis. 
A  considerable  proportion  of  the  cases  returned  from 
the  Philippine  Islands  during  1900  and  1901,  and 
probably  since  then,  diagnosed  as  amoebic  dysentery, 


86  PARASITIC  AMCEB.E  OF  MAN. 

were  really  cases  of  enteritis  showing  Entamoeba  coll 
in  the  feces.  Aside  from  the  zoological  standpoint, 
it  is  of  great  practical  importance  that  we  recognize 
the  fact  that  this  non-pathogenic  species  occurs  both 
in  health  and  disease  in  the  intestine  of  man  and  that 
we  be  able  to  differentiate  between  Entamoeba  coli 
and  the  pathogenic  entamcebse.  This  is  often  a  very 
difficult  matter  when  the  organisms  are  few  in  num- 
ber, and  at  certain  stages  of  development,  while  it  is 
always  true  that  considerable  experience  in  the  ex- 
amination of  the  feces  is  required  before  such  a 
differentiation  can  be  easily  made.  The  exceedingly 
superficial  study  that  many  practitioners  make  of 
these  parasites  can  never  result  in  success  in  the  dif- 
ferentiation of  species. 

MORPHOLOGY  IN  FRESH  AND  STAINED  PREPARA- 
TIONS.— To  secure  living  specimens  of  Entamaeba  coli 
it  is  always  best  to  administer  an  ounce  of  magnesium 
sulphate  or  a  Seidlitz  powder  before  examining  the 
feces,  as  this  washes  off  the  amoebae  from  the  mucous 
membrane  and  greatly  facilitates  their  demonstration. 
The  technique  employed  in  preparing  such  specimens 
for  examination  has  already  been  considered. 

Entamoeba  coli  consists  of  a  mass  of  cytoplasm 
containing  a  well-defined  nucleus,  and  in  a  few  in- 
stances, one  or  more  vacuoles.  The  differentiation 
between  the  ectoplasm  and  the  endoplasm  is  not  well 


8 


13 


16 


FIG. II.— Entamceba  coli.  (After  Casagrandi  and  Barbagallo.)  15, 13, 10, 
and  7,  various  stages  in  nuclear  division  of  Entamceba  coli;  4  and  1,  divi- 
sion of  the  nucleus  into  two  as  shown  in  the  vegetative  stage  of  growth;  16  and 
11,  schizogony  and  the  formation  of  eight  daughter-nuclei  during  the  vegeta- 
tive stage  of  development;  8,  expulsion  of  a  portion  of  the  nucleus  prior  to 
encystment;  5,  first  stage  of  encystment  showing  the  homogeneous  cyto- 
plasm; 2,  oval  encysted  form  with  homogeneous  cytoplasm;  17,  encysted 
form  within  which  eight  daughter-nuclei  have  developed;  14,  12,  and  9, 
illustrating  various  stages  of  nuclear  division  in  the  encysted  forms;  6,  two 
young  amoebae  leaving  the  cyst  in  which  they  have  developed;  3,  two  young 
amoeba?  just  after  leaving  the  parent  cyst. 


AMCEB.E  OF  THE  INTESTINAL  TRACT.       87 

marked,  although  the  cytoplasm  is  thus  divided. 
Motility  is  present,  but  is  very  sluggish  as  compared 
to  Entamceba  Mstolytica.  Under  favorable  condi- 
tions reproduction  occurs  by  simple  division  and 
schizogony,  with  the  production  of  eight  daughter 
amoebaB.  If  unfavorable  conditions  arise  the  organ- 
isms encyst  and  eight  daughter  cells  are  formed  within 
the  cyst,  which  are  liberated  under  favorable  con- 
ditions. The  morphology  of  the  parasite  will  be 
considered  in  detail,  but  it  should  be  remembered  that 
individual  organisms  vary  greatly  in  appearance  at 
different  stages  of  development  and  that  the  de- 
scription given  is  a  composite  picture  which  is  true 
of  the  vast  majority  of  parasites  belonging  to  this 
species. 

Size. — As  a  rule,  Entamceba  coli  is  not  as  large  as 
Entamceba  Iwistolytica,  although  individuals  are  often 
observed  which  approximate  the  size  of  the  latter  or- 
ganism. The  measurements  given  by  different 
authorities  vary  greatly,  as  one  would  expect,  for 
during  the  different  stages  of  development  the  para- 
sites may  measure  from  5  microns  to  50  microns  in 
diameter,  and  as  every  specimen  of  feces  will  contain 
organisms  in  different  developmental  stages  the  varia- 
tions in  size  reported  do  not  indicate  inaccuracy  on  the 
part  of  the  observer.  Schaudinn  states  that  Enta- 
mceba  coli  varies  between  8  and  50  microns  in 


88  PARASITIC  AMGEBJE  OF  MAN. 

diameter,  but  in  my  experience  it  is  very  rarely  that 
one  of  these  organisms  exceeds  25  to  30  microns  in 
diameter.  It  may  be  stated,  as  a  general  rule,  that 
this  parasite  measures  from  10  to  20  microns  in  diam- 
eter, while  the  average  measurement  of  Entamceba 
Mstolytica  is  from  25  to  35  microns. 

The  difference  in  size  between  the  two  species  is 
of  but  slight  value  in  differentiation  and  a  separation 
of  the  amoebae  of  the  human  intestine  into  species, 
based  upon  the  size  of  the  organism,  is  both  unscien- 
tific and  erroneous.  It  is  one  of  the  least  valuable  of 
our  methods  of  differentiation. 

Some  authorities  have  stated  that  pathogenic 
amoebae  are  always  larger  than  Entamceba  coli,  but 
I  have  very  frequently  observed  cases  of  pure 
infection  with  Entamceba  Mstolytica  in  which  the 
amoebae  were  as  small  as  the  ordinary  Entamceba  coli. 
As  a  matter  of  fact,  both  large  and  small  amoebae  are 
present  in  every  infection,  so  that  it  is  impossible  to 
differentiate  between  them  on  account  of  their  size. 

During  the  encysted  stage  of  development  En- 
tamceba coli  is  much  smaller  than  during  its  vege- 
tative stage,  the  average  diameter  being  from  10  to 
15  microns. 

Shape. — The  shape  varies  with  the  motility  of  the 
organism.  When  motionless  Entamceba  coli  is  spher- 
ical or  slightly  oval  in  shape,  but  when  in  motion  the 


AMOEBJE  OF  THE  INTESTINAL  TRACT.       89 

shape  continually  changes,  depending  upon  the  con- 
tour of  the  pseudopodia,  but  the  latter  are  always 
rounded,  and  never  spinose,  as  are  the  pseudopodia 
of  many  of  the  amoebae  cultivated  from  external 
sources.  The  writer  has  never  observed  an  amoeba 
in  the  feces  having  spinose  pseudopodia,  although 
free-living  forms  must  frequently  occur  in  the  feces, 
especially  in  tropical  countries,  but  probably  in  the 
encysted  stage. 

The  shape  of  the  pseudopodia  is  useful  in  differ- 
entiating this  species  from  the  pathogenic  amoebae, 
such  as  Entamoeba  Tustolytica  and  Entamoeba  tetra- 
gena.  In  the  latter  parasites  the  pseudopodia  are 
generally  finger-like  in  shape  and  of  considerable  size, 
while  in  Entamceba  coli  they  are  short  and  blunt. 
Aside  from  this  characteristic  of  the  pseudopodia,  the 
shape  of  the  organisms  is  of  no  value  in  the  differen- 
tiation of  species. 

Color. — Entamoeba  coli  may  be  said  to  be  of  a 
peculiar  dull  grayish  color,  and  this  is  of  some  value 
in  the  differentiation  of  the  species,  the  pathogenic 
amoebae  of  the  intestine  appearing  slightly  greenish 
in  color  or  almost  colorless.  Musgrave  and  Clegg  be- 
lieve that  color  simply  means  environment,  the  green- 
ish color  being  due  to  the  absorption  of  blood  serum 
containing  haemoglobin,  and  that  in  feces  in  which 
blood  is  not  present  the  amoebae  never  present  this 


90  PARASITIC  AMOEBA  OF  MAN. 

color.  While  this  may  be  the  reason  for  the  greenish 
coloration  frequently  observed  in  Entamceba  histolyt- 
ica,  it  certainly  does  not  apply  in  the  case  of  En- 
tamceba  coli,  for  the  addition  of  blood  to  the  feces 
containing  this  organism  does  not  result  in  the  ab- 
sorption of  either  dissolved  haemoglobin  or  the 
phagocytosis  of  red  blood  corpuscles. 

It  is  extremely  difficult  to  describe  the  exact  color 
of  this  parasite,  but  to  one  who  has  studied  the  various 
species  of  amoebae  the  difference  in  the  coloring  of 
Entamceba  coli  and  the  pathogenic  amoebae  is  quite 
characteristic. 

Cytoplasm. — The  cytoplasm  of  Entamceba  coli  is 
divided  into  an  ectoplasm  and  an  endoplasm.  These 
two  portions  of  the  cytoplasm  are  exceedingly  difficult 
to  distinguish  in  all  stages  of  the  growth  of  the  para- 
site, and  impossible  unless  the  organism  is  in  motion, 
and  in  many  specimens  the  ectoplasm  cannot  be  dis- 
tinguished from  the  endoplasm  even  when  the  parasite 
is  actively  motile.  Schaudinn  first  called  attention  to 
this  point  and  to  the  fact  that  the  ectoplasm  of  En- 
tamoeba  coli  is  much  less  refractive  to  light  than  is 
the  endoplasm,  in  those  instances  in  which  the  two 
portions  can  be  distinguished. 

The  ectoplasm  presents  a  homogeneous  appear- 
ance and  unless  a  high  power  lens  is  used  no  definite 
structure  can  be  distinguished ;  with  an  immersion  lens 


AMCEB.E  OF  THE  INTESTINAL  TRACT.       91 

it  appears  to  be  composed  of  very  minute  granules. 
When  the  parasite  encysts,  the  ectoplasm  is  replaced 
by  a  refractive,  smooth,  or  slightly  wrinkled  cyst  wall, 
impervious  to  staining  solutions  and  apparently  of 
very  dense  structure.  In  some  instances  definite 
layers  of  a  very  refractive  material  compose  the  wall 
of  the  cyst,  but  generally  it  has  a  distinct  double 
outline. 

The  endoplasm,  constituting  the  greater  portion 
of  the  cytoplasm,  is  of  a  finely  granular  structure  and 
an  examination  with  a  high  power  lens  shows  it  to  be 
composed  of  a  delicate  network  enclosing  a  fluid 
medium  containing  multitudes  of  fine  granules.  In 
most  amoebae  numerous  bacteria  are  observed  in  the 
endoplasm,  as  well  as  crystals  derived  from  the  feces, 
and  in  some  organisms  one,  or  perhaps  two,  small 
vacuoles.  During  reproduction  by  schizogony  the 
endoplasm  contains  from  2  to  8  oval,  slightly  refrac- 
tive bodies,  which  may  be  mistaken  for  vacuoles,  but 
which  are  really  the  daughter  amoebae.  The  mor- 
phology of  the  parasite  during  this  stage  of  develop- 
ment has  led  to  the  erroneous  statement  that  Enta- 
mceba  coli  contains  numerous  vacuoles. 

During  the  encysted  stage  the  endoplasm  is 
homogeneous  in  appearance  and  contains  from  2  to 
8  round,  refractive  bodies,  representing  the  daughter 
amoeba?. 


92  PARASITIC  AMOEBAE  OF  MAN. 

The  entire  cytoplasm  in  the  vast  majority  of  Ent- 
amceba  coli  appears  to  be  composed  of  endoplasm,  as 
the  differentiation  of  the  ectoplasm  is  impossible  in 
most  instances.  In  the  very  young  amoebae  of  this 
species  the  cytoplasm  is  homogeneous  throughout. 

During  motion  the  pseudopodia,  which  are 
formed  by  the  ectoplasm,  appear  to  be  somewhat  less 
refractive  than  the  endoplasm,  and  of  very  slight 
consistence.  Sometimes  the  pseudopodia  are  only 
visible  upon  careful  focussing,  resembling  a  veil-like 
membrane  projecting  from  some  portion  of  the  pe- 
riphery of  the  parasite. 

Vacuoles  and  Contained  Bodies. — The  vast  ma- 
jority of  parasites  belonging  to  this  species  present 
a  finely  granular  cytoplasm  in  which  no  vacuoles  can 
be  observed.  In  the  thousands  of  specimens  studied 
by  myself  a  vacuole  was  present  in  but  a  little  over 
10  per  cent,  of  the  parasites  and  a  very  small  per- 
centage showed  more  than  one  vacuole.  When  pres- 
ent the  vacuole  is  of  small  size,  and  never  contractile. 
The  absence  of  vacuoles  in  this  species  is  in  striking 
contrast  to  the  numerous  vacuoles  observed  in  Enta- 
mceba  histolytica,  and  other  pathogenic  amoebae,  and 
is  of  some  value  in  the  differentiation  of  this  species. 

The  endoplasm  contains  refractive  dots  and  rods, 
some  of  which  are  micrococci,  bacilli,  and  various 
crystals  derived  from  the  feces,  while  the  nature  of 


AMCEB^E  OF  THE  INTESTINAL  TRACT.       93 

others  is  still  undetermined.  These  bodies  are  better 
differentiated  in  stained  preparations. 

Red  blood  corpuscles  are  rarely  observed  in  the 
cytoplasm  of  Entamceba  coll,  and  this  point  is  of 
some  value  in  the  differentiation  of  the  species.  When 
present,  the  cytoplasm  never  contains  more  than  one 
or  two,  and  I  believe  that  they  are  of  purely  accidental 
occurrence  and  that  normally  this  parasite  is  not 
phagocytic  for  red  blood  corpuscles.  Experimentally 
it  is  almost  impossible  to  make  these  parasites  engulf 
the  erythrocytes  when  blood  is  added  to  feces  con- 
taining them,  although  in  the  case  of  Entamoeba 
histolytica,  the  engulfing  of  these  cells  occurs  very 
frequently. 

The  development  of  the  daughter-nuclei  during 
schizogony  has  already  been  noted.  These  bodies  ap- 
pear within  the  cytoplasm  as  oval,  slightly  refractive 
areas,  measuring  from  3  to  5  microns  in  diameter 
and  lying  within  the  endoplasm.  They  may  be  easily 
mistaken  for  vacuoles  by  one  untrained  in  the  study 
of  amoebae.  In  stained  specimens  these  bodies  take 
the  chromatin  stain. 

Nucleus. — The  most  prominent  body  in  the  cyto- 
plasm of  Entamoeba  coll  is  the  nucleus,  which  is 
almost  always  distinctly  visible,  thus  differentiating 
this  parasite  from  Entamoeba  histolytica,  in  which 
the  nucleus  is  generally  invisible. 


94  PARASITIC  AMOEBAE  OF  MAN. 

If  one  desires  to  study  the  changes  occurring  in 
the  nucleus  during  reproduction  the  best  results  are 
obtained  with  special  staining  methods,  and  by  the 
use  of  a  warm-stage  or  an  incubator  in  which  the 
microscope  can  be  placed  during  the  examination. 
It  is  possible  to  observe  division  of  the  nucleus  in 
the  living  parasite  by  the  use  of  the  warm-stage  or 
incubator,  but  patience  is  required  in  order  to  demon- 
strate the  changes  which  occur. 

The  size  of  the  nucleus  varies,  but  it  generally 
measures  from  5  to  8  microns  in  diameter.  During 
certain  stages  of  division  the  nucleus  may  be  either 
larger  or  smaller  than  the  average  given,  and  during 
this  period  it  may  be  hard  to  distinguish.  In  most 
instances  the  shape  of  the  nucleus  is  spherical,  but 
sometimes  it  is  distinctly  oval  in  contour.  It  is 
bounded  by  a  well  defined,  heavy,  nuclear  membrane 
which  in  the  living  organism  appears  highly  refractile. 
Upon  the  inner  surface  of  this  membrane  there  occur 
brightly  refractile  elevations,  generally  hemispherical 
in  shape,  consisting  of  nuclear  chroma  tin,  and  dots 
and  irregular  granules  of  the  same  substance  may  be 
observed  scattered  throughout  the  nucleus.  At  or 
near  the  centre  of  the  nucleus,  during  the  vegetative 
stage  of  existence,  there  are  generally  observed  from 
one  to  two  very  distinct  masses  of  chromatin  forming 
the  karyosome.  The  substance  of  the  nucleus,  aside 


AMOEBAE  OF  THE  INTESTINAL  TRACT.       95 

from  the  chromatin,  is  apparently  composed  of  fluid 
material  enclosed  in  a  very  delicate  achromatic  net- 
work. 

When  the  organisms  are  in  motion  the  nucleus 
tends  to  retain  its  position  near  the  centre,  although 
at  times  it  may  be  observed  at  the  periphery  of  the 
moving  parasite. 

In  some  instances  the  limiting  membrane  of  the 
nucleus  is  very  thick  and  perfectly  smooth  in  out- 
line, while  the  chromatin  is  collected  in  irregular 
masses  within  the  membrane,  the  karyosome  being 
indistinguishable.  This  appearance  is  especially 
noticeable  just  prior  to  encystment  and  does  not  occur 
during  the  vegetative  stage  of  existence.  Again, 
amoeba?  are  observed  in  which  the  chromatin  is 
arranged  in  very  irregular  masses  upon  the  inner 
surface  of  the  nuclear  membrane  or  close  to  it,  while 
the  karyosome  is  well  differentiated.  Many  different 
appearances  as  regards  the  situation  of  the  chromatin 
are  presented  by  the  nucleus  in  various  stages  of 
development,  but  the  most  common  are  those  which 
have  been  described. 

Stained  Preparations. — The  methods  which  may 
be  used  for  staining  Entamceba  coli  have  already  been 
described,  but  I  have  found  that  Wright's  method 
and  iron  hsematoxylin  give  the  best  results.  I  have 
observed  but  little  difference  in  the  results  obtained 


96  PARASITIC  AMOEBAE  OF  MAN. 

with  either  wet  or  dry-fixed  preparations,  when 
Wright's  stain  is  used,  but  if  iron  hsematoxylin  be 
used  the  wet-fixed  preparations  give  the  best  results 
as  regards  the  structure  of  the  nucleus.  Some  author- 
ities have  claimed  that  unless  the  preparations  are 
wet-fixed  no  deductions  can  be  drawn  regarding  the 
structure  of  the  nucleus  in  stained  preparations,  but 
with  this  statement  I  must  entirely  disagree.  It 
makes  but  little  difference  whether  the  specimens  be 
fixed  wet  or  dry,  when  Wright's  stain  or  any  other 
modification  of  the  Romano wsky  stain  is  used,  although 
the  best  results  are  obtained  in  wet-fixed  preparations. 
I  cannot  but  believe  that  many  of  the  appearances 
presented  in  wet-fixed  preparations  are  just  as  arti- 
ficial as  any  that  may  be  observed  in  the  dry-fixed 
preparations,  and  there  is  considerable  reason  for 
concluding  that  both  wet-  and  dry-fixed  preparations 
should  be  used  in  the  study  of  this  class  of  protozoa. 
When  Wright's  stain  is  used  Entamceba  coli 
presents  three  distinct  portions,  the  ectoplasm,  the 
endoplasm  and  the  nucleus.  However,  it  should  be 
remembered  that  these  three  divisions  are  not  always 
visible,  as  not  infrequently  the  entire  amoeba  stains 
uniformly  throughout,  with  the  exception  of  the  nu- 
cleus. The  period  of  development  apparently  makes 
a  great  difference  in  the  facility  with  which  the  or- 
ganism takes  the  stain,  as  well  as  with  the  reaction 


AMCEB^E  OF  THE  INTESTINAL  TRACT.       97 

to  the  stain  employed,  and  in  every  preparation  it 
will  be  found  that  the  majority  of  the  parasites  stain 
but  poorly,  and  that,  as  a  rule,  a  large  number  of 
smears  will  have  to  be  stained  and  examined  before 
a  good  preparation  will  be  found.  If  one  attempts 
to  follow  the  life  cycle  of  the  parasite  in  stained 
preparations  it  will  be  necessary  to  examine  scores 
of  smears  before  each  step  can  be  demonstrated,  but 
if  one  has  the  patience  it  is  possible  to  follow  almost 
every  stage  of  development  in  such  preparations. 

In  those  organisms  in  which  the  ectoplasm  and 
the  endoplasm  are  differentiated  it  is  observed  that 
the  ectoplasm  stains  a  very  delicate  blue  and  appears 
almost  structureless,  while  the  endoplasm  stains  a 
dark  blue  or  violet,  and  is  composed  of  deep  blue 
granules  and  irregular  masses  of  homogeneous  ma- 
terial. The  distinction  between  the  light  blue  ecto- 
plasm and  deep  blue  or  violet  endoplasm  is  very 
marked  and  serves  to  differentiate  this  organism  from 
Entamceba  liistolytica,  in  which  the  ectoplasm  takes 
a  deep  blue,  and  the  endoplasm  a  light  blue  color. 

If  one  examines  the  well-stained  organisms  with 
a  high  power  lens  the  ectoplasm  will  seem  to  be  com- 
posed of  very  minute,  dust-like,  dimly  stained  gran- 
ules, while  the  endoplasm  contains  deeply  stained 
bacteria  of  various  kinds  and  sometimes  a  few  crys- 
tals. If  vacuoles  are  present  they  are  observed  as  un- 


98  PARASITIC  AMCEEM  OF  MAN. 

stained  spherical  or  oval  areas  within  the  endoplasm. 
With  the  Wright  stain  the  nuclear  chromatin 
takes  a  bright  red  or  crimson  color  and  the  nuclear 
membrane  stains  very  deeply,  the  chromatin  situated 
upon  its  inner  surface  appearing  as  bright  red  or 
crimson  masses.  When  over-stained  the  entire  nucleus 
is  bright  red  and  no  morphological  details  can  be 
recognized.  Within  the  substance  of  the  nucleus 
the  chromatin  may  be  observed  as  delicate  threads, 
rods,  or  granules,  stained  a  bright  red  in  color  and 
separated  by  unstained  spaces.  The  karyosome 
stains  a  deep  red  or  violet  and  is  usually  well  differen- 
tiated. Just  prior  to  division,  during  the  vegetative 
stage,  the  nuclear  chromatin  may  be  observed  as  two 
crimson  stained  masses  lying  at  the  poles  of  the 
elongated  nucleus,  but  with  this  stain  I  have  never 
observed  distinct  evidences  of  mitosis.  In  specimens 
stained  with  the  iron-hasmatoxylin  method  evidence 
of  a  primitive  mitosis  are  sometimes  observed.  The 
extrusion  of  the  chromatin  from  the  nucleus  during 
certain  stages  of  reproduction,  first  described  by 
Schaudinn,  is  often  evidenced  in  stained  preparation 
by  the  presence  of  minute  deeply  stained  granules 
of  chromatin  lying  free  in  the  endoplasm.  The  ap- 
pearance of  the  nucleus  in  stained  preparations  dur- 
ing the  various  reproductive  stages  will  be  found 
described  in  the  section  dealing  with  this  subject. 


AMCEBJE  OF  THE  INTESTINAL  TRACT.       99 

Motility. — Entamoeba  coli  may  be  described  as  a 
sluggishly  motile  amoeba  in  which  this  property  is 
often  absent,  and  when  present,  is  of  slight  duration 
and  very  limited  in  extent.  Motility  is  made  possible 
by  the  extrusion  of  pseudopodia  composed  of  ecto- 
plasm, the  character  of  which  has  already  been  de- 
scribed, but  it  may  be  recalled  that  they  are  small  and 
rounded  in  contour  and  less  refractile  to  light  than 
is  the  endoplasm,  while  in  many  instances  it  is  im- 
possible to  distinguish  the  boundary  between  the  pseu- 
dopodium  and  the  endoplasm,  even  when  the  organism 
is  moving.  Motility  is  always  most  marked  in  freshly 
voided  f  eces  and  is  seldom  observed  in  f  eces  which  has 
stood  at  room  temperature  for  more  than  one  hour. 

Two  forms  of  motility  are  frequently  observed; 
the  first  consisting  in  the  extrusion  of  pseudopodia 
into  which  flows  the  endoplasm,  thus  producing  a  very 
sluggish  progressive  motion;  the  second,  consisting 
in  the  extrusion  of  pseudopodia  from  different  por- 
tions of  the  periphery  at  the  same  time,  thus  causing 
a  change  in  the  shape  of  the  organism,  but  no  pro- 
gressive motion. 

The  sluggish  motility  of  this  species  of  amoeba 
when  compared  with  the  active  motility  of  such  species 
as  Entamoeba  lustolytica  and  Entamoeba  tetragena 
is  of  considerable  value  in  differentiation,  for  one 
never  observes  in  Entamoeba  coli  a  progressive  motion 


100  PARASITIC  AMOEBJS  OF  MAN. 

which  can  be  compared  in  activity  with  that  com- 
monly observed  in  the  two  other  species  mentioned. 
Reproduction. — Before  Schaudinn's  researches 
little  was  known  regarding  the  exact  methods  of 
reproduction  of  the  amoebae  occurring  in  the  human 
intestine.  A  few  observers  had  roughly  described 
certain  reproductive  phenomena,  but  as  they  were 
unable  to  distinguish  species  their  descriptions  are 
confused  and  of  little  value  in  the  study  of  this  sub- 
ject. Celli  and  Fiocca,  working  in  all  probability 
with  Entamceba  coli,  described  the  life  cycle  as  con- 
sisting of  an  amoeboid  stage,  a  resting  stage  and  an 
encysted  stage,  but  their  descriptions  are  incomplete, 
and  it  was  not  until  the  work  of  Casagrandi  and 
Barbagallo  appeared  that  the  exact  method  of  repro- 
duction in  Entamceba  coli  was  known.  They  described 
simple  division  and  the  formation,  under  certain  con- 
ditions, of  cysts  in  which  eight  young  amoebae  devel- 
oped. Their  work  was  confirmed  and  extended  by 
Schaudinn,  who  found  that  reproduction  during  the 
vegetative  stage  occurs  by  simple  division  and  by 
schizogony,  with  the  formation  of  eight  daughter 
amoebae.  When  conditions  are  unfavorable  for  vege- 
tative existence  cysts  are  formed  in  which  eight  young 
amoebae  are  developed.  Complicated  nuclear  changes 
occur  during  schizogony  and  development  within  the 
cysts,  which  are  best  studied  in  preparations  kept 


FIG.  V. —  Photomicrograph  of  Entamceba  coli  and  Entamceba  histo- 
lytica. (After  Jiirgens.)  The  amceba  at  1  is  Entamceba  coli,  while  the 
other  amoebae  in  the  photograph  are  all  examples  of  Entamceba  histolytica. 
Note  the  well-defined  nucleus  in  Entamceba  coli  and  the  absence  of  a  distinct 
nucleus  in  Entamceba  histolytica.  Note  the  clear  ectoplasm  in  Entamceba 
histolytica. 


FIG.  VI. —  Multiplication  by  simple  division  in  Entamceba  coli.  (Cfraig.) 
A,  division  of  the  chromatin  within  the  nucleus  into  several  clumps  arranged 
upon  the  inside  of  the  nuclear  membrane;  B,  formation  of  a  nuclear  spindle, 
showing  that  the  division  of  the  nucleus  is  mitotic  in  character;  C  and  Z>, 
division  of  the  nucleus  into  two  portions;  E,  division  of  the  nucleus  and 
partial  division  of  the  cytoplasm  of  the  amoeba;  F,  complete  division  of  the 
parasite  and  the  production  of  two  amoebae. 


AMCEBJS  OF  THE  INTESTINAL  TRACT.     101 

upon  a  warm-stage  or  under  the  incubator-micro- 
scope or  in  wet-fixed  preparations  stained  with  iron 
haematoxylin.  I  have  been  able  to  follow  almost  every 
developmental  stage  as  described  by  Schaudinn  and 
the  following  description  of  reproduction  in  Enta- 
mceba  coli  is  based  upon  that  given  by  Schaudinn  and 
upon  my  own  observations. 

In  reproduction  by  simple  division,  which  is  not 
infrequently  observed  in  the  feces,  the  following 
phenomena  may  be  demonstrated  in  the  living 
organism:  the  nucleus  elongates,  the  nuclear  mem- 
brane becoming  thinner  and  less  refractile  while  the 
chromatin,  which  is  visible  as  brightly  refractile  dots 
or  granules,  becomes  concentrated  at  each  pole  of  the 
nucleus.  After  the  nucleus  has  become  much 
elongated  a  constriction  develops  near  the  centre  and 
finally  division  occurs,  two  nuclei  being  produced. 
Coincident  with  the  elongation  and  division  of  the 
nucleus  the  protoplasm  of  the  amoeba  becomes  less 
granular  and  a  constriction  appears  which  deepens 
and  finally  becomes  complete  after  the  division  of 
the  nucleus.  In  this  way  two  amcebse  are  formed  and 
in  stained  specimens  the  division  of  the  chromatin  and 
the  nuclear  changes  can  be  followed,  the  chromatin 
staining  a  bright  red  with  Wright's  stain.  If  iron 
haematoxylin  be  used,  after  wet-fixation,  clear  evi- 
dences of  mitosis  are  frequently  observed. 


102  PARASITIC  AMCEB^E  OF  MAN. 

In  reproduction  by  schizogony  complicated  nu- 
clear changes  occur  resulting  in  the  formation  of  eight 
daughter  cells.  The  beginning  of  the  process  is  first 
evidenced  by  swelling  of  the  nucleus  apparently  due 
to  the  absorption  of  fluids  from  the  endoplasm,  while 
at  the  same  time  the  latter  extrudes  all  foreign  ma- 
terial, and  the  entire  organism  becomes  motionless. 
The  chromatin  situated  upon  the  inner  surface  of  the 
nuclear  membrane  divides  into  eight  little  heaps  which 
finally  become  free  within  the  nucleus,  and  this  is 
followed  by  the  rupture  of  the  nuclear  membrane 
and  the  liberation  of  the  eight  daughter  nuclei  within 
the  endoplasm.  After  this  occurs  the  cytoplasm  of 
the  amoeba  divides  into  eight  more  or  less  irregular 
portions  each  containing  one  of  the  daughter  nuclei 
and  in  this  way  eight  young  amoebae  are  produced. 
This  process  was  first  studied  by  Casagrandi  and 
Barbagallo  and  the  plate  reproduced  from  their  work 
well  illustrates  the  daughter  nuclei  immediately  after 
liberation  from  the  nucleus. 

In  the  living  specimen  the  daughter  nuclei  are 
visible  as  brightly  ref ractile  masses  of  granules  due  to 
the  chromatin  of  which  they  are  largely  composed, 
while  in  stained  specimens  the  chromatin  takes  a 
bright  red  color  when  Wright's  method  is  employed, 
so  that  this  method  of  reproduction  can  be  easily 
traced  in  such  preparations. 


AMOEBA  OF  THE  INTESTINAL  TRACT.     103 

Reproduction  within  a  cyst  occurs  when  conditions 
become  unfavorable  to  vegetative  existence.  In 
freshly  voided  specimens  of  f eces  this  process  is  very 
rarely  observed,  but  in  specimens  which  have  been 
kept  for  some  time  this  method  of  reproduction  may 
be  easily  studied.  If  the  infection  has  existed  for 
some  time  encysted  forms  are  more  frequently  ob- 
served in  the  freshly  voided  feces,  for,  like  many 
other  protozoan  organisms,  these  parasites  multiply 
for  a  long  period  of  time  asexually,  but  after  a  cer- 
tain number  of  generations  the  process  of  reproduc- 
tion assumes  a  sexual  character,  self-fertilization 
occurring  within  a  cyst. 

The  cysts  of  Entamoeba  coli  are  spherical  or  oval 
bodies  measuring  from  10  to  15  microns  in  diameter, 
as  a  rule.  The  appearance  of  the  cyst  wall  varies  in 
different  stages  of  development,  but  it  is  always  re- 
fractile  and  generally  presents  a  double  outline.  A 
mammillated  cyst  wall  is  sometimes  noted,  but  is  by  no 
means  commonly  observed. 

Just  before  encystment  the  amoeba  becomes  per- 
fectly motionless,  while  the  endoplasm  clears  itself  of 
granular  material  and  all  foreign  particles.  This 
process  reduces  the  size  of  the  organism  about  one- 
third,  and  after  it  is  complete  there  forms  upon  the 
surface  a  very  refractile,  delicate,  hyaline  membrane 
which  appears  to  be  formed  from  the  substance  of 


104  PARASITIC  AMGEBJE  OF  MAN. 

the  ectoplasm.  At  first  this  membrane  appears  single 
in  outline,  but  as  reproduction  proceeds  a  double  out- 
line develops  and  in  some  instances  a  well  defined  mam- 
millation  of  the  outer  surface  of  the  cyst  wall  occurs. 

The  cytoplasm,  after  the  formation  of  the  cyst 
wall,  is  perfectly  hyaline  in  appearance  and  contains  a 
nucleus  in  which  the  chromatin  appears  to  have 
greatly  increased  in  amount.  If  the  cysts  are  placed 
under  conditions  favorable  to  development  reproduc- 
tive changes  occur  in  the  nucleus  which  may  be  briefly 
described  as  follows: 

The  nucleus  first  divides  by  a  primitive  mitosis 
into  two  daughter  nuclei  of  the  same  size.  These 
two  nuclei  move  to  opposite  sides  of  the  cyst  and  the 
cytoplasm  gathers  around  them,  dividing  the  organ- 
ism into  two  partly  separated  portions.  In  the  living 
specimen  the  nuclei  are  observed  to  disappear  after 
several  hours,  but  if  stained  specimens  be  studied  it 
will  be  noted  that  the  chromatin  of  the  nuclei  is  very 
largely  distributed  to  the  cytoplasm,  although  a 
definite  portion  of  the  nucleus  remains,  and  from  this 
a  new  nucleus  is  formed.  If  living  specimens  be 
watched  it  will  be  observed  that  after  the  disappear- 
ance of  the  nuclei  they  will  again  reappear,  but  con- 
tain much  less  chromatin  and  are  of  smaller  size. 

Schaudinn  describes  certain  variations  in  the 
primary  changes  occurring  in  the  nucleus  as  follows : 


FIG.  VII. —  Schizogonyof.  Entamceba  coli.  (Craig.)  A,  division  of 
the  nuclear  chromatin  into  eight  distinct  masses  arranged  upon  the  inside 
of  the  nuclear  membrane;  B,  division  of  the  nucleus  into  eight  daughter- 
nuclei;  C,  further  stage  of  the  same  process  of  division;  D,  complete  division 
of  the  amoeba  into  eight  daughter  amcebse. 


FIG.  VIII. —  Sporogony  of  Entamceba  coli.  (Craig.)  A,  an  amoeba 
ridding  itself  of  extraneous  matter  just  prior  to  encystment;  B,  division  of 
the  nucleus  into  two  daughter-nuclei;  C,  division  of  the  daughter-nuclei  into 
two  nuclei;  D,  eight  nuclear  stage  of  the  cyst  of  Entamceba  coli;  E,  F,  and 
G,  various  stages  in  the  sporogony  of  Entamceba  coli;  II ,  fully  developed 
cyst  of  Entamceba  coli,  containing  eight  daughter-nuclei;  /,  the  young 
amcebse  as  they  appear  after  liberation  from  the  parent  cyst. 


AMOEBA  OF  THE  INTESTINAL  TRACT.     105 

1.  The  daughter  nuclei  may  dissolve  entirely,  the 
plasma    being  filled   with   chromatin   granules   and 
cords,  most  of  which  disappear,  while  out  of  one  part 
the  new  nuclei  are  reconstructed. 

2.  The  daughter  nuclei  dissolve  with  the  exception 
of    a    small    central    portion,    while    the    chromatic 
granules  and  cords  are  partly  expelled  and  partly 
resorbed. 

3.  The     daughter     nuclei     extrude     chromatic 
granules  and  cords  to  the  plasma,  but  remain  dis- 
tinctly visible  and  are  finally  expelled  from  the  cyst. 
In  this  case  the  new  nuclei  are  formed  from  the  free 
chromatin  particles. 

Other  variations  are  noted,  but  are  not  of  suffi- 
cient interest  to  be  described.  Schaudinn  believes  that 
the  manner  of  reconstruction  of  the  nucleus  depends 
upon  the  degree  of  development  of  the  cyst  wall. 
He  says: 

"  My  observations  showed  to  me  that  the  indicated 
variability  is  connected  with  the  degree  of  develop- 
ment of  the  cystic  sheath.  For  if  the  reconstruction 
of  the  nuclei  ensues  while  the  gelatin  sheath  is  only 
very  weakly  developed,  or  not  at  all,  the  casting  out 
of  the  perishing  parts  of  the  nucleus  is  possible;  but 
if  the  gelatin  sheath  is  strongly  developed  or  the  dif- 
ferentiation of  the  real  cystic  membrane  has  begun, 
then  the  perishing  substances  remain  in  the  plasma 


106  PARASITIC  AMCEB^E  OF  MAN. 

and  a  complete  dissolution  of  the  nuclei  takes  place." 

After  the  reconstruction  of  the  daughter  nuclei 
they  are  situated  at  opposite  poles  of  the  cyst  and  the 
following  changes  may  then  be  observed: 

Both  divide  by  primitive  mitosis  so  that  two  nuclei 
lie  at  the  opposite  poles.  One  of  these  contracts, 
forming  a  reduction  body,  while  the  other  again 
divides.  This  results  in  the  presence  of  three  nuclei  at 
each  pole,  two  of  them  normal  in  appearance,  while 
one  is  degenerating.  One  of  the  newly  formed  nuclei 
also  degenerates  eventually,  leaving  two  nuclei  at 
each  pole  of  the  cyst. 

After  this  has  occurred,  the  body  of  the  amoeba 
contracts  still  further  and  the  cystic  membrane  be- 
comes more  pronounced,  appearing  firmer  and  more 
refractile*  The  division  between  the  two  portions  of 
cytoplasm  is  lost,  the  two  pair  of  nuclei  divide  mitot- 
ically,  two  daughter  nuclei  being  formed,  called  the 
active  and  passive  pronuclei.  These  pronuclei  fuse, 
forming  two  synkarya,  each  of  which  divides  into  four, 
so  that  the  cyst  now  contains  eight  daughter  nuclei. 
During  this  time  the  walls  of  the  cyst  have  become 
firmer  and  thicker  and  there  is  no  indication  of 
division  of  the  cytoplasm.  Under  favorable  condi- 
tions, such  as  reception  into  a  new  host,  the  cyst  wall 
dissolves,  the  cytoplasm  divides  into  eight  irregular 
masses  each  containing  a  daughter  nucleus,  and  in 


AMOEBA  OF  THE  INTESTINAL  TRACT.     107 

this  way  eight  young  amoebae  are  produced.  Schau- 
dinn  states  that  of  the  cysts  voided  with  the  feces 
only  from  10  to  20  per  cent,  undergo  reproduction 
in  the  manner  described,  and  this  agrees  with  my  own 
observations.  Many  preparations  have  to  be  studied 
before  the  entire  process  of  reproduction  can  be  fol- 
lowed, but  sometimes  one  is  so  fortunate  as  to  be  able 
to  follow  the  entire  development  in  a  single  individual. 

ERRATUM 

The  paragraph  upon  page  107  beginning  "  The  process  of 
reproduction  within  a  cyst,  etc.,"  should  be  omitted,  as  it 
relates  to  Entamoeba  histolytica,  and  should  follow  the  last 
paragraph  upon  page  140. 


CULTIVATION. — I  believe  that  it  may  be  stated 
with  truth  that  Entamoeba  coli  has  never  been  arti- 
ficially cultivated.  I  have  used  all  the  methods  which 
have  been  recommended  for  this  purpose,  but  with- 
out success. 

RELATION  TO  DISEASE. — The  wide  distribution  of 
this  parasite  in  both  healthy  individuals  and  those 
suffering  from  diseases  other  than  dysentery;  the  fact 
that  scores  of  cases  have  been  observed  in  which  the 
parasites  have  been  constantly  present  for  months  and 
even  years  without  producing  symptoms  of  diarrhoea 
or  dysentery;  and  the  negative  results  obtained  by 
animal  experiments,  all  definitely  prove  that  Enta- 
mceba  coli  is  not  a  pathogenic  species.  It  may  be  of 


106  PARASITIC  AMCEBJS  OF  MAN. 

and  a  complete  dissolution  of  the  nuclei  takes  place. " 

After  the  reconstruction  of  the  daughter  nuclei 
they  are  situated  at  opposite  poles  of  the  cyst  and  the 
following  changes  may  then  be  observed: 

Both  divide  by  primitive  mitosis  so  that  two  nuclei 
lie  at  the  opposite  poles.  One  of  these  contracts, 
forming  a  reduction  body,  while  the  other  again 
divides.  This  results  in  the  presence  of  three  nuclei  at 


sTlll  lurther  and  tne  cystic  membrane  be- 
comes more  pronounced,  appearing  firmer  and  more 
refractile.  The  division  between  the  two  portions  of 
cytoplasm  is  lost,  the  two  pair  of  nuclei  divide  mitot- 
ically,  two  daughter  nuclei  being  formed,  called  the 
active  and  passive  pronuclei.  These  pronuclei  fuse, 
forming  two  synkarya,  each  of  which  divides  into  four, 
so  that  the  cyst  now  contains  eight  daughter  nuclei. 
During  this  time  the  walls  of  the  cyst  have  become 
firmer  and  thicker  and  there  is  no  indication  of 
division  of  the  cytoplasm.  Under  favorable  condi- 
tions, such  as  reception  into  a  new  host,  the  cyst  wall 
dissolves,  the  cytoplasm  divides  into  eight  irregular 
masses  each  containing  a  daughter  nucleus,  and  in 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     107 

this  way  eight  young  amoeba?  are  produced.  Schau- 
dinn  states  that  of  the  cysts  voided  with  the  feces 
only  from  10  to  20  per  cent,  undergo  reproduction 
in  the  manner  described,  and  this  agrees  with  my  own 
observations.  Many  preparations  have  to  be  studied 
before  the  entire  process  of  reproduction  can  be  fol- 
lowed, but  sometimes  one  is  so  fortunate  as  to  be  able 
to  follow  the  entire  development  in  a  single  individual. 

The  process  of  reproduction  within  a  cyst  has  to 
be  studied  from  living  preparations  entirely,  as  I  have 
not  found  any  method  of  staining  the  cysts  nor  have  I 
been  successful  with  any  method  recommended  by 
other  observers. 

CULTIVATION. — I  believe  that  it  may  be  stated 
with  truth  that  Entamoeba  coli  has  never  been  arti- 
ficially cultivated.  I  have  used  all  the  methods  which 
have  been  recommended  for  this  purpose,  but  with- 
out success. 

RELATION  TO  DISEASE. — The  wide  distribution  of 
this  parasite  in  both  healthy  individuals  and  those 
suffering  from  diseases  other  than  dysentery;  the  fact 
that  scores  of  cases  have  been  observed  in  which  the 
parasites  have  been  constantly  present  for  months  and 
even  years  without  producing  symptoms  of  diarrhoea 
or  dysentery;  and  the  negative  results  obtained  by 
animal  experiments,  all  definitely  prove  that  Enta- 
moeba coli  is  not  a  pathogenic  species.  It  may  be  of 


108  PARASITIC  AMOEBAE  OF  MAN. 

interest  to  review  briefly  the  experimental  work  re- 
lating to  the  pathogenicity  of  Entamceba  coli.  Kar- 
tulis  experimented  with  amoebae  obtained  from 
healthy  individuals  by  injecting  them  into  the  intes- 
tine of  cats,  but  was  not  able  to  produce  any  patho- 
logical lesions  or  symptoms  of  disease.  The  same 
negative  results  were  obtained  by  Kruse  and  Pas- 
quale.  One  of  the  strongest  arguments  used  by  Celli 
and  Fiocca  against  the  pathogenic  action  of  amoebae 
was  the  fact  that  they  were  not  able  to  produce  dysen- 
tery in  cats  by  the  injection  of  amoebae  from  healthy 
individuals,  and  the  very  careful  experiments  of 
Kovacs  were  equally  unsuccessful.  Strong  and  Mus- 
grave  were  also  unable  to  produce  dysentery  in  cats 
with  the  amoebae  found  in  health,  and  their  comment 
regarding  their  negative  results  is  of  interest.  They 
say  "  one  of  these  cases  which  has  been  under  our  ob- 
servation for  several  months  has  had  these  harmless 
amoebae  in  his  stools  constantly  during  that  time,  yet 
he  has  no  dysentery  and  no  history  of  any,  and  he 
has  no  intestinal  trouble.  We  have  repeatedly  in- 
jected large  numbers  of  these  non-dysenteric  amoebae 
(amoeba  coli)  while  motile  in  the  stools,  into  the 
rectum  of  cats,  but  with  no  eff ect.  We  have  neither 
been  able  to  produce  dysentery  with  them  nor  any 
lesions  of  the  large  bowel — on  the  other  hand  we 
have  had  no  difficulty  in  producing  dysentery  and 


AMCEB^E  OF  THE  INTESTINAL  TRACT.     109 

ulcerations  of  the  large  bowel  in  cats  by  the  injection 
of  the  stools  or  contents  of  liver  abscesses  containing 
motile  amoebse  dysenteric." 

The  work  of  Jiirgens  is  of  special  interest  in  con- 
nection with  Entamceba  coli.  He  demonstrated  that 
this  parasite  is  not  able  to  penetrate  the  normal  mu- 
cous membrane  of  the  intestine  because  of  the  slight 
strength  of  the  pseudopodia  and  his  observations  have 
been  confirmed  by  Schaudinn.  The  latter  observer 
was  unable  to  produce  dysentery  or  any  lesion  of  the 
bowel  in  animals  by  feeding  experiments  or  the  rectal 
injection  of  material  containing  Entamceba  coU.  He 
was  successful  in  infecting  young  cats  and  many  of 
his  studies  upon  reproduction  were  made  on  amoebse 
obtained  from  such  animals,  but  the  infection  never 
resulted  in  either  symptoms  or  lesions  of  dysentery. 
He  twice  infected  himself  by  swallowing  encysted 
forms,  in  both  instances  the  experiment  being  con- 
trolled by  regular  examinations  of  his  feces  for  two 
months  before  the  experiment.  After  swallowing  the 
cysts  numerous  amoebse  appeared  in  his  feces,  but  in 
neither  instance  did  any  symptoms  of  diarrhoea  or 
dysentery  develop  and  the  amoebae  gradually  disap- 
peared. 

My  personal  observations  cover  a  large  number  of 
experiments  in  which  young  kittens  were  used  as  the 
animals  experimented  upon.  I  have  injected  into 


110  PARASITIC  AMGEBJE  OF  MAN. 

the  rectum  of  kittens  fecal  material  containing  both 
encysted  and  motile  forms  of  Entamceba  coli  and 
have  never  been  able  to  produce  the  least  symptom 
of  diarrhoea  or  dysentery,  although  50  per  cent,  of 
kittens  injected  with  material  containing  Entamosba 
Mstolytica  developed  dysentery,  of  which  many  of 
them  died.  I  have  repeated  these  injections  upon 
the  same  animal  from  5  to  10  times  and  in  no  case 
was  any  evidence  of  intestinal  inflammation  produced. 

I  have  made  many  feeding  experiments  in  which 
kittens  were  given  milk  containing  multitudes  of 
encysted  and  motile  forms  of  Entamceba  coli  and  in 
not  a  single  instance  were  any  symptoms  of  diarrhoea 
or  dysentery  produced,  although  the  feedings  were 
repeated  at  frequent  intervals.  In  kittens  fed  with 
milk  containing  Entamceba  histolytica,  on  the  other 
hand,  over  65  per  cent,  developed  severe  dysentery. 

To  one  who  has  carefully  followed  the  growth  of 
our  knowledge  concerning  the  amoebge  of  man  it  is 
impossible  to  doubt  that  Entamceba  coli  is  a  harmless 
commensal  occurring  in  a  very  large  percentage  of 
healthy  individuals  and  in  individuals  suffering  from 
diseases  in  which  inflammatory  conditions  of  the  in- 
testine can  be  excluded.  Were  this  parasite  the  cause 
of  a  form  of  dysentery,  as  is  still  believed  by  some, 
practically  50  per  cent,  of  individuals  in  nearly  every 
locality  would  suffer  from  this  disease.  All  of  the 


AMOEBAE  OF  THE  INTESTINAL  TRACT.     Ill 

arguments  which  have  been  brought  forward  in  the 
endeavor  to  show  that  this  species  is  simply  a  non- 
virulent  form  of  the  amoebae  commonly  found  in 
dysentery,  have  one  by  one  been  abandoned  in  the 
face  of  the  evidence  which  has  accumulated  as  to  the 
specific  nature  of  the  parasite. 

The  recognition  of  this  species  is  of  practical 
importance  because  many  patients  have  been  diag- 
nosed as  suffering  from  amoebic  dysentery  upon  the 
mere  presence  of  Entamceba  coli  in  the  feces.  I  have 
known  patients  to  be  treated  with  rectal  injections 
for  weeks  in  whom  the  only  evidence  of  dysentery 
was  the  presence  of  amoebae  in  the  stools,  which  upon 
careful  examination  proved  to  be  Entamceba  coli.  It 
is  undoubtedly  true  that  the  statistics  of  the  Army 
regarding  the  occurrence  of  this  disease  in  the  Philip- 
pines are  greatly  vitiated  by  records  of  diagnoses 
based  upon  the  mere  presence  of  amoebae  in  the  feces, 
no  effort  having  been  made  to  differentiate  the 
species.  It  must  be  admitted  that  such  differentiation 
cannot  be  made  by  the  tyro  in  this  work,  but  trained 
laboratory  assistance  is  generally  available  in  most 
localities  and  a  correct  diagnosis  is  of  enough  im- 
portance to  warrant  submitting  material  to  an  expert. 
It  is  just  as  reasonable  to  advocate  treating  every 
case  of  fever  with  quinine  on  the  supposition  that  the 
malarial  plasmodia  are  present,  as  it  is  to  treat  all 


PARASITIC  AMOEBAE  OF  MAN. 

individuals  showing  amoebae  in  the  stools  for  amoebic 
dysentery.  This  advice  has  been  given  by  some 
authorities,  but  if  it  were  followed  out,  over  one-half 
of  the  population  of  many  tropical  localities  would  be 
under  treatment  for  amoebic  dysentery.  This  is  well 
illustrated  by  the  observations  of  Ashburn  and  my- 
self in  Manila,  P.  I.,  where  we  found  over  72  per 
cent,  of  the  Hospital  Corps  men  on  duty  at  the 
Division  hospital  infected  with  Entamceba  coli.  It 
is  not  difficult  to  imagine  the  condition  in  this  hos- 
pital had  we  advocated  placing  these  men  under  treat- 
ment, and  yet  this  is  just  what  is  recommended  by 
those  who  refuse  to  accept  Schaudinn's  classification. 
The  fact  that  it  is  difficult  in  many  instances  to  dif- 
ferentiate the  non-pathogenic  species  from  the  patho- 
genic is  no  excuse  for  failure  to  do  so,  for  so  much 
depends  upon  the  diagnosis  of  amoebic  dysentery  that 
it  should  never  be  made  unless  the  pathogenic  species 
has  been  demonstrated  in  the  feces. 

The  reports  made  by  a  few  observers,  notably 
Musgrave,  of  severe  lesions  of  dysentery  being  found 
at  autopsy,  although  no  symptoms  of  the  disease  had 
ever  been  present,  must  be  doubted.  I  have  autop- 
sied  hundreds  of  cases  of  amoebic  dysentery  and  I 
have  never  yet  observed  a  case  in  which  well  marked 
lesions  were  present  but  that  a  history  of  attacks  of 
diarrhoea  or  dysentery  could  be  obtained.  It  is  im- 


AMOEBAE  OF  THE  INTESTINAL  TRACT.     113 

possible  to  believe  that  marked  lesions  of  this  disease 
can  occur  in  an  intestine  without  leading  to  the  pro- 
duction of  clinical  symptoms.  It  is  possible  that  in 
isolated  instances  a  few  ulcers  might  exist  in  limited 
regions  of  the  intestine  without  producing  symptoms 
which  would  attract  the  attention  of  the  patient,  but 
that  any  generalized  ulceration  can  occur  without 
symptoms  I  am  not  ready  to  believe.  The  lack  of  a 
history  of  diarrhoea  or  dysentery  cannot  be  depended 
upon  in  native  races,  for  such  symptoms  are  not  con- 
sidered of  enough  importance  by  these  people  to  be 
noted.  Almost  all  of  Musgrave's  cases  were  in  Fili- 
pinos who  suffer  continually  from  diarrhoea  and  who 
would  not  be  apt  to  notice  a  symptom  which  is  more 
or  less  constantly  present.  We  have  no  record  of 
such,  cases  occurring  in  temperate  regions  among 
people  in  whom  the  symptoms  would  be  apt  to  attract 
attention. 

However,  the  occurrence  of  the  lesions  of  amoebic 
dysentery  in  patients  who  have  not  suffered  from 
symptoms  of  that  disease  is  of  no  value  as  an  argu- 
ment against  the  specificity  of  Entamceba  coli,  and 
I  believe  there  is  sufficient  evidence  at  hand  to  prove 
conclusively  that  this  species  is  valid  and  that  it  occurs 
commonly  as  a  harmless  commensal  in  the  human  in- 
testine. 


114  PARASITIC  AMOEBJE  OF  MAN. 

ENTAMCEBA  HISTOLYT1CA.     Schaudinn,  1903. 

This  species  was  first  differentiated  by  Schaudinn 
in  1903,  although  it  had  been  previously  studied  in 
a  thorough  manner  by  Kartulis,  Jiirgens,  Council- 
man and  Lafleur,  Strong  and  Musgrave,  and  many 
others.  It  is  a  pathogenic  species,  causing  amoebic 
dysentery,  its  distribution  probably  being  world-wide, 
although  it  occurs  most  frequently  in  tropical  and 
sub-tropical  regions.  The  pathological  lesions 
produced  by  it,  as  well  as  the  experimental  evidence, 
prove  conclusively  that  it  is  the  cause  of  a  distinct 
form  of  dysentery  which  is  endemic  in  many  localities 
and  which  may  become  epidemic  when  conditions  are 
favorable. 

GEOGRAPHICAL  DISTRIBUTION. — This  species  of 
amoebae  has  been  demonstrated  in  the  Philippine 
Islands,  by  Ashburn  and  myself,  Vedder,  and  other 
observers;  in  Formosa,  by  Nakagawa;  in  Cochin 
China,  by  Pfuhl  and  other  French  investigators;  in 
Siam,  by  Wooley;  in  India,  by  Fearnside,  Powell, 
Rogers,  Viereck  and  Duncan  and  Anderson;  in 
Africa,  by  Kartulis,  Marchoux,  Huge,  A.  Plehn, 
Wellman,  and  Prout;  in  Europe,  especially  in  Aus- 
tria and  Poland,  by  Hlava  and  many  other  observers; 
in  South  America,  by  Dessy  and  Marotta;  and  in 
the  United  States,  by  Osier,  Musser,  Dock,  Ellis, 
Tuttle,  Boggs,  Stockton,  Patterson  and  others. 


AMCEB^E  OF  THE  INTESTINAL  TRACT.     115 

In  a  collective  study  of  the  occurrence  of  amoebic 
dysentery  in  the  United  States,  Patterson  records 
cases  reported  from  Maine,  New  Hampshire,  New 
York,  Pennsylvania,  Maryland,  District  of  Columbia, 
Virginia,  West  Virginia,  North  Carolina,  South 
Carolina,  Georgia,  Florida,  Tennessee,  Alabama, 
Mississippi,  Ohio,  Illinois,  Missouri,  Michigan, 
Minnesota,  Montana,  Arkansas,  Indian  Territory 
and  Texas.  Long  has  reported  numerous  cases 
originating  in  California  and  it  is  very  probable  that 
in  this  State  the  infection  was  imported  by  soldiers 
returning  from  the  Philippine  Islands. 

While  the  disease  is  wide-spread  in  the  United 
States  and  is  probably  much  more  common  than  is 
generally  supposed,  it  has  nowhere  been  reported  in 
epidemic  form,  but  occurs  as  isolated  cases  in  many 
of  which  the  exact  origin  of  the  infection  is  prob- 
lematical. Entamoeba  histolytica  is  generally  consid- 
ered as  the  exciting  cause  in  the  infections  reported 
as  originating  in  this  country,  but  unfortunately  the 
species  present  have  not  been  studied  thoroughly 
and  it  may  be  that  many  of  these  cases  are  really  due 
to  a  distinct  species  endemic  in  the  United  States. 
I  have  recently  found  Entamoeba  tetragena  in  cases 
originating  in  the  United  States  as  well  as  Enta- 
moeba Mstolytica. 

MORPHOLOGY. — Like  Entamoeba  coli  this  parasite 


116  PARASITIC  AMOEBAE  OF  MAN. 

consists  of  a  mass  of  cytoplasm  containing  a  nucleus 
and  varying  in  shape  when  in  motion,  although  it  is 
always  spherical  or  oval  in  outline  when  motionless. 
The  cytoplasm  contains  a  nucleus  and  one  or  more 
vacuoles  which  are  not  contractile.  The  cytoplasm 
is  divided  into  two  distinct  portions,  the  ectoplasm 
and  the  endoplasm,  the  ectoplasm  being  clear  and 
glass-like  in  appearance,  while  the  endoplasm  is  less 
refractile  and  more  granular  in  structure.  The  endo- 
plasm contains  the  nucleus,  vacuoles,  and  may  con- 
tain red  blood  corpuscles,  bacteria,  crystals,  or  other 
material  which  may  have  been  engulfed  by  the  para- 
site. The  nucleus  is  generally  invisible  and  contains 
a  very  minute  karyosome.  Movement  is  accom- 
plished by  the  extension  and  retraction  of  very 
distinct  pseudopodia  formed  by  the  ectoplasm.  Re- 
production occurs  by  simple  division  and  by  sporula- 
tion  or  gemmation. 

The  following  detailed  description  of  the  mor- 
phology of  this  parasite  is  the  result  of  careful  study 
of  many  thousand  of  these  organisms  and  refers  only 
to  the  morphology  of  the  amoebse  as  they  are  observed 
in  the  feces.  I  have  not  been  able  to  obtain  cultural 
forms  of  this  parasite,  although  some  authorities  have 
claimed  to  have  done  so. 

Size. — The  size  of  Entamoeba  histolytica  is 
variously  stated  by  different  observers,  but  it  may 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     117 

be  said  that  it  generally  measures  from  25  to  50 
microns  in  diameter.  In  the  vast  majority  of  in- 
stances this  parasite  is  considerably  larger  than  Enta- 
mceba  coli,  but,  of  course,  the  size  varies  with  the 
stage  of  development.  However,  I  am  of  the  opinion 
that  the  size  is  generally  under-  rather  than  over- 
stated by  most  writers.  From  my  own  observations 
I  believe  that  Entamceba  histolytica,  during  its  vege- 
tative stage,  is  seldom  less  than  15  microns  in 
diameter,  and  generally  much  larger.  If  we  remem- 
ber that  the  average  blood  corpuscle  measures  7 
microns  in  diameter  it  will  be  at  once  apparent  that 
we  seldom  see  amoebae  in  cases  of  dysentery  which 
are  as  small  as  one  or  two  of  these  cells.  It  is  also 
well  known  that  this  species  is  phagocytic  for  red 
blood  corpuscles  and  it  is  not  at  all  unusual  to  see 
from  3  to  6  of  these  cells  within  the  small  amoebae.  I 
have  observed  amoeba?  of  this  species  containing  from 
10  to  30  red  blood  corpuscles  and  this  gives  a  definite 
idea  of  the  large  size  of  some  of  these  parasites.  A 
measurement  of  50  microns  in  diameter  is  not  infre- 
quently observed  and  the  vast  majority  of  these  para- 
sites measure  from  30  to  35  microns,  a  much  greater 
average  measurement  than  that  of  Entamoeba  coll. 

These  remarks  do  not  apply  to  the  encysted  stage 
of  development  or  to  the  young  spores.  The  cysts 
measure  from  10  to  20  microns  in  diameter,  while  the 
spores  average  about  5  microns  in  diameter. 


118  PARASITIC  AMOEBAE  OF  MAN. 

The  size  of  this  species  is  of  some  value  in  differen- 
tiating it  from  other  amoebae,  especially  Entamoeba 
coli,  but  it  cannot  be  depended  upon  alone.  Large 
and  small  amoeba?  occur  in  almost  every  specimen  of 
feces  examined,  so  that  a  classification  based  upon 
size  alone  must  be  erroneous.  Some  investigators 
have  endeavored  to  make  the  distinction  into  patho- 
genic and  non-pathogenic  amoebse  rest  simply  upon 
the  size  of  the  organism,  the  larger  organisms  being 
classed  as  pathogenic,  and  the  smaller  as  non-patho- 
genic. From  my  experience  I  cannot  agree  with 
these  conclusions.  Careful  examination  of  the  feces 
of  cases  of  amoebic  dysentery  often  show  that  amoebae 
of  large  size  are  not  always  present,  while  in  other 
cases  both  large  and  small  amoebae  belonging  to  this 
species  are  found.  I  am  free  to  confess  that  as  a  rule 
the  majority  of  the  organisms  are  approximately  of 
the  same  size  in  most  specimens  of  feces,  but  this  does 
not  prove  anything  more  than  that  they  are  nearly  all 
in  the  same  stage  of  development.  I  have  repeatedly 
observed  cases  of  dysentery  in  which  the  vast  ma- 
jority of  the  amoebae  present  in  the  feces  were  much 
smaller  than  the  average  given  for  Entamoeba  histo- 
lytica,  but  in  which  the  clinical  symptoms  were  the 
same  as  in  the  cases  where  the  larger  amoebse  were 
found,  while  the  morphology  of  the  organisms  was 
identical  with  that  of  Entamoeba  histolytica. 


AMOEBAE  OF  THE  INTESTINAL  TRACT.     119 

The  size  of  this  parasite  is  of  importance  in  diag- 
nosis as  there  is  no  parasite  which  has  been  so  fre- 
quently mistaken  for  epithelial  cells  or  for  leucocytes 
when  in  the  non-motile  condition.  When  moving  the 
organism  is  easily  recognized,  but  when  non-motile  it 
is  most  difficult  to  differentiate  from  other^  cells  oc- 
curring in  the  feces.  In  fact  it  has  been  stated  by 
some  good  observers  that  it  is  not  safe  to  diagnose  the 
presence  of  amoebae  in  feces  unless  motility  is  ob- 
served, but  while  this  may  be  good  advice  for  the 
beginner,  it  is  too  general  a  statement,  for  if  the 
organisms  are  not  undergoing  degeneration  they  are 
easily  recognized  even  when  non-motile  by  one  who  has 
had  experience  in  the  study  of  this  class  of  parasites. 

If  it  is  remembered  that  these  organisms  are 
larger,  as  a  rule,  than  the  leucocytes  or  intestinal 
epithelial  cells ;  that  they  generally  contain  red  blood 
cells;  and  are  divided  into  two  distinct  portions,  an 
ectoplasm  and  endoplasm;  it  should  not  be  difficult 
to  differentiate  them  from  other  bodies  occurring  in 
the  feces. 

Shape. — When  resting,  Entamoeba  histolytica  is 
generally  spherical  in  shape,  although  it  is  not  un- 
usual to  observe  oval  organisms.  When  in  motion 
great  variation  in  shape  is  observed,  due  to  the  pseu- 
dopodia,  so  that  it  is  impossible  to  accurately  describe 
the  contour  of  the  parasite  at  this  time. 


120  PARASITIC  AMOEBJE  OF  MAN. 

Color. — I  have  already  called  attention  to  the  dull 
grayish  color  of  Entamceba  coli  and  to  the  lack  of 
differentiation  between  the  ectoplasm  and  the  endo- 
plasm.  In  Entamceba  Mstolytica  the  ectoplasm  is 
almost  colorless  and  very  ref ractile,  resembling  a  piece 
of  ground  white  glass,  while  the  endoplasm  is  of  a 
very  light  gray  color,  in  many  instances  tinged  with 
green,  due  to  absorbed  haemoglobin.  In  cases  in 
which  the  stools  contain  blood,  the  majority  of  the 
amoeba?  observed  show  this  greenish  tint  due,  I  be- 
lieve, to  haemoglobin  liberated  during  the  digestion  of 
the  red  blood  corpuscles.  That  this  is  true  can  be  ex- 
perimentally demonstrated  if  one  cares  to  spend  the 
time  to  do  so.  I  have  many  times  observed  an  amoeba 
cross  the  microscopic  field  and  engulf  a  red  blood  cor- 
puscle and  have  watched  the  latter  slowly  disappear, 
the  endoplasm  at  the  same  time  assuming  a  greenish 
color. 

It  is  very  common  to  observe  not  only  well  pre- 
served red  blood  cells  within  this  organism,  but  also 
fragmented  corpuscles,  and  in  such  instances  it  is 
always  noted  that  the  endoplasm  is  distinctly  green 
in  color.  This  phenomenon  proves  that  Entamceba 
Mstolytica  is  capable  of  engulfing  and  destroying  the 
red  blood  corpuscles  of  its  host  and  serves  to  dis- 
tinguish it  from  Entamceba  coli,  for  in  the  few  in- 
stances in  which  I  have  observed  red  blood  corpuscles 


AMCEBJE  OF  THE  INTESTINAL  TRACT. 

within  the  latter  organism,  I  have  never  seen  any 
evidence  indicating  that  the  amoebae  were  able  to 
digest  them.  I  believe  that  in  Entamceba  coli  the 
engulfing  of  red  corpsucles  is  purely  accidental,  and 
that  the  cells  are  extruded  without  being  digested. 

The  Protoplasm. — The  appearance  of  the  proto- 
plasm varies  considerably  with  the  age  of  the  para- 
site. In  the  small  or  young  amoebse,  the  protoplasm 
is  finely  granular  in  appearance  and  the  ectoplasm 
and  endoplasm  can  seldom  be  distinguished  unless  the 
organism  is  in  motion,  but  in  the  larger  and  older 
parasites  this  distinction  can  frequently  be  made  even 
when  motility  is  absent.  The  protoplasm  contains  a 
nucleus  situated  to  one  side  of  the  centre  of  the  para- 
site, which  is  generally  invisible.  One  or  more 
vacuoles  are  present,  as  well  as  crystals,  bacteria, 
pigment  granules  and  amorphous  material. 

The  Cytoplasm. — In  the  fully  developed  parasite 
the  cytoplasm  is  divided  into  two  distinct  portions, 
the  ectoplasm  and  the  endoplasm.  The  ectoplasm 
comprises  about  one-third  of  the  cytoplasm  and  is 
perfectly  hyaline  and  glass-like  in  appearance.  If  a 
high  power  lens  is  used  it  appears  to  be  composed  of 
dense  material  containing  innumerable  very  minute 
granules.  It  is  very  refractile,  much  more  so  than 
the  endoplasm,  and  is  easily  recognized.  The  ecto- 
plasm gives  one  the  impression  of  a  firm  structure 


PARASITIC  AMCEB^E  OF  MAN. 

capable  of  penetrating  soft  tissues  and  Schaudinn 
claims  that  it  is  owing  to  this  property  that  these 
organisms  push  their  way  into  the  intestinal  wall.  He 
says: 

"  The  harmless  Ent amoeba  coU  with  its  soft  pseu- 
dopodia  is  unable  to  penetrate  the  healthy  epithelial 
layer  of  the  intestine,  while  the  dysentery  amoeba  by 
means  of  its  tough  ectoplasm  can  do  so.  This  is 
easily  observable  upon  fresh  sections  of  infected  cat 
intestines  in  which  the  amoebae  will  crawl  about  for 
hours,  forcing  the  cells  of  the  epithelium  asunder, 
thus  working  their  way  into  the  tissues." 

One  has  but  to  compare  the  firm  appearance  and 
well  defined  ectoplasm  of  Entamceba  histolytica  with 
the  delicate  ectoplasm  of  Entamceba  coli  to  be  con- 
vinced of  the  truth  of  Schaudinn's  arid  Jiirgens's  as- 
sertion that  the  secret  of  the  pathogenic  action  of 
Entamceba  histolytica  lies  partly  in  the  ability  of  the 
ectoplasm  to  penetrate  the  mucous  membrane  of  the 
intestine,  a  property  not  possessed  by  the  ectoplasm 
of  Entamceba  coli  because  of  its  delicate  structure. 

The  endoplasm,  which  comprises  about  two-thirds 
of  the  body  of  the  amoeba,  is  light  grayish  in  color 
and  is  composed  of  granular  material  enclosed  within 
an  ill-defined  reticular  structure.  It  is  much  coarser 
than  the  ectoplasm  and  less  refractile. 

Inclosed  within  the  endoplasm  there  are  generally 


AMOEBA  OF  THE  INTESTINAL  TRACT. 

one  or  more  vacuoles,  non-contractile  in  character.  In 
some  of  the  larger  parasites  these  vacuoles  are  very 
numerous,  and  such  organisms  are  probably  degen- 
erating. Besides  the  vacuoles,  small  oval  bodies  are 
often  observed,  the  nature  of  which  is  still  in  doubt, 
although  they  are  generally  interpreted  as  being  the 
nuclear  portion  of  the  spores  which  bud  from  the 
organism.  Red  blood  corpuscles,  crystals,  and  bac- 
teria are  generally  observed  lying  within  the  endo- 
plasm. 

The  Nucleus. — It  is  generally  difficult  to  dis- 
tinguish a  nucleus  in  this  species,  a  fact  which  serves 
to  assist  in  differentiating  it  from  Entamceba  coti,  the 
nucleus  in  the  latter  being  visible  in  nearly  every 
organism  as  a  distinct  body  possessing  a  well  marked 
nuclear  membrane,  masses  of  chromatin  and  a  definite 
karyosome.  The  nucleus  of  Entamceba  histolytica  is 
generally  situated  excentrically  in  the  endoplasm, 
often  near  the  boundary  of  the  ecto-  and  the  endo- 
plasm, or  it  may  be  in  contact  with  the  border  of  the 
ectoplasm,  being  flattened  out  against-  it.  The  size 
varies  with  the  stage  of  growth  of  the  parasite,  but  it 
averages  about  5  microns  in  diameter.  When  the 
organism  is  moving  the  nucleus  continually  changes 
its  position  and  it  is  exceedingly  difficult  to  study  it  at 
this  time,  owing  to  its  continual  disappearing  from 
view  within  the  endoplasm. 


PARASITIC  AMGEBJE  OF  MAN. 

A  well  defined  nuclear  membrane  cannot  be  dis- 
tinguished, the  border  of  the  nucleus  being  of  the 
same  refraction  as  the  hyaloplasm.  By  very  careful 
focussing  it  is  sometimes  possible  to  demonstrate  a 
very  delicate  limiting  membrane,  slightly  more  re- 
fractile  than  the  rest  of  the  nucleus. 

The  amount  of  chromatin  contained  within  the 
nucleus  of  Entamoeba  histolytica  is  much  less  than 
in  the  nucleus  of  Entamoeba  coli.  A  small  chromatic 
zone  is  frequently  present  at  the  border  of  the  nucleus 
and  a  few  minute  granules  are  sometimes  observed 
within  the  hyaloplasm,  but  this  is  in  marked  con- 
trast to  the  nucleus  of  Entamoeba  coli,  in  which  the 
chromatin  is  distinctly  visible,  both  as  well  defined 
elevations  upon  the  inner  surface  of  the  thick  nucleus 
membrane  and  as  masses  of  granules  within  the  hyalo- 
plasm. The  relative  proportion  of  chromatin  and  its 
arrangement  is  well  shown  in  the  specimens  stained 
by  Wright's  method,  which  will  be  described  later. 

A  karyosome  is  situated  near  the  centre  of  the 
nucleus  and  is  very  small  and  delicate  in  appearance. 

The  nucleus  is  most  easily  observed  in  young 
amoebae  and  the  reason  for  this  is  demonstrated  in 
stained  specimens  where  it  is  noted  that  in  the  larger 
amoebas  the  chromatin  of  the  nucleus  is  distributed  to 
the  cytoplasm,  the  remainder  of  the  nucleus  forming 
a  residual  body,  irregular  in  shape,  which  it  would 


AMOEBA  OF  THE  INTESTINAL  TRACT.     125 

be  very  difficult,  if  not  impossible,  to  demonstrate  in 
the  living  specimen.  During  reproduction  by  gemma- 
tion the  nucleus  is  seldom  visible  in  the  living  prepara- 
tion, but  during  simple  division  it  can  generally  be 
distinguished,  lying  near  the  centre  of  the  parasite. 

The  characteristic  features  of  the  nucleus  of  Enta- 
inceba  Mstolytica  are  its  lack  of  a  well  defined  nuclear 
membrane,  the  small  amount  of  nuclear  chromatin,  the 
minute  karyosome,  often  invisible,  and  the  fact  that 
it  is  generally  invisible  or  distinguished  with  great 
difficulty. 

Vacuoles  and  Contained  Bodies. — The  endoplasm 
of  this  species  generally  contains  one  or  more  vacuoles 
which  are  not  contractile.  In  the  young  parasites  a 
vacuole  may  be  absent,  but  generally  a  single  one  is 
present,  while  in  the  fully  developed  organisms  from 
one  to  ten  or  even  more  may  be  present,  the  average 
number  being  two  or  three.  If  only  one  vacuole  be 
present  it  is  of  large  size  and  spherical  in  shape,  but 
if  they  are  multiple  the  size  varies  and  some  of  them 
appear  oval  in  outline. 

In  many  instances  the  vacuoles  appear  to  contain 
bacteria,  haemoglobin,  small  refractile  granules,  or 
other  material,  suggesting  that  they  are  digestive  in 
character,  but  I  have  never  observed  any  evidence 
that  they  are  contractile.  When  the  organisms  are 
in  motion  the  vacuoles  are  continually  changing  their 
position  within  the  endoplasm. 


126  PARASITIC  AMCEBJS  OF  MAN. 

In  some  amoebse  the  vacuoles  comprise  nearly  all 
of  the  substance  of  the  organism,  the  ectoplasm  being 
invisible,  while  the  endoplasm  consists  merely  of  a 
network  enclosing  the  numerous  vacuoles.  Such 
amoeba?  are  undoubtedly  undergoing  degeneration, 
for  though  they  may  retain  their  motility  for  some 
time,  it  will  be  observed  they  eventually  undergo 
fragmentation.  The  significance  of  the  vacuoles  in 
Entamceba  histolytica  is  still  uncertain.  The  pres- 
ence of  various  substances  within  them  suggests  that 
they  have  something  to  do  with  digestion,  but  the 
absence  of  contractility  places  them  apart  from  the 
class  of  contractile  vacuoles  which  are  present  in 
many  free-living  amoebae.  However,  it  is  probable 
that  they  have  a  distinct  function,  although  when 
they  are  present  in  large  numbers  I  believe  that  they 
are  due  to  degeneration  of  the  cytoplasm. 

One  of  the  most  characteristic  biological  features 
of  Entamceba  Mstolytica  is  its  power  of  phagocyting 
the  red  blood  corpuscles  of  its  host.  This  property 
can  be  easily  demonstrated  by  adding  fresh  blood  to 
specimens  of  feces  containing  motile  amoebae,  and 
there  can  be  no  doubt  but  that  the  red  corpuscles  are 
digested  within  the  parasites.  These  cells  are  very 
commonly  observed  within  this  species,  not  only  when 
the  feces  contain  blood,  but  often  in  cases  where  there 
is  no  macroscopic  evidence  of  its  presence.  The 


AMCEB^E  OF  THE  INTESTINAL  TRACT.     127 

number  of  red  cells  which  may  be  contained  within  an 
amoeba  is  sometimes  enormous,  and  I  have  often  ob- 
served organisms  so  filled  with  these  cells  that  very 
little  of  the  structure  could  be  distinguished. 

In  cases  of  amoebic  dysentery  in  which  both  Enta- 
mceba  coli  and  Entamceba  Mstolytica  are  present 
it  is  but  seldom  that  the  former  contain  red  blood 
corpuscles  while  the  majority  of  the  latter  may  be 
filled  with  them.  As  a  rule  only  two  to  six  red  cells 
are  contained  within  an  amoeba,  and  if  the  process  of 
digestion  is  to  be  studied  an  organism  should  be 
selected  which  contains  from  one  to  two  red  cor- 
puscles. If  such  organisms  are  kept  at  body  tem- 
perature the  red  cells  will  be  seen  to  gradually  break 
up,  the  haemoglobin  being  liberated,  imparting  to  the 
endoplasm  a  greenish  color.  Extrusion  of  the  red 
cell  is  sometimes  observed,  but  this  is  an  abnormal 
process  due  to  unfavorable  environment,  except  in 
those  organisms  in  which  reproductive  changes  are 
occurring. 

In  many  amoebae  the  red  corpuscles  become  de- 
colorized, only  a  delicate  shadow  of  the  cell  remain- 
ing visible.  Such  decolorized  corpuscles  are  often 
mistaken  for  small  vacuoles,  but  careful  focussing  will 
reveal  their  nature.  These  shadow-corpuscles  must 
also  be  differentiated  from  the  refractile  oval  bodies 
to  be  mentioned. 


128  PARASITIC  AMOEBA  OF  MAN. 

Besides  the  vacuoles  and  red  corpuscles  the  endo- 
plasm  generally  contains  pigment  granules,  crystals 
of  various  kinds,  and  bacteria.  In  addition  there 
occur  small  oval  bodies  giving  the  reaction  of 
chromatin  when  stained,  which  form  the  chromatic 
portion  of  the  nucleus  of  young  amoebse  which  are 
developing  within  the  parent  organism. 

Motility. — In  fresh  specimens  of  feces  Entamceba 
histolytica  is   actively  motile,   much  more   so  than 
Entamceba  coll.    This  property  is  rendered  possible 
by  the  pseudopodia,  which  in  this  species  are  well 
differentiated,  appearing  clear,  refractile,  and  firm 
in  consistence.    To  study  the  production  of  the  pseu- 
dopodia it  is  necessary  to  keep  the  specimen  at  body 
temperature,  and  if  this  is  done  motility  is  main- 
tained for  hours.    Even  in  specimens  of  feces  kept 
at  room  temperature  the  motility  of  this  parasite  is 
often  retained  for  a  long  time,  motile  amoeba?  being 
sometimes  observed  after  from  two  to  six  hours.  The 
rate  of  motility  is  of  some  value  in  differentiating  this 
species   from  Entamceba  coli,  in  which  motility  is 
always  feeble.     The  mere  extrusion  of  pseudopodia 
is  more  rapid,  even  though  progressive  motion  may 
not  occur,  while  the  division  between  the  ectoplasm 
and  endoplasm  is  very  distinct. 

The  shape  of  the  pseudopodia  is  of  importance 
in  the  diagnosis  of  this  organism.     It  will  be  re- 


AMGEB^E  OF  THE  INTESTINAL  TRACT.     129 

membered  that  in  the  Entamoeba  coll  the  pseudopodia 
are  short  and  blunt,  but  in  this  species  they  are 
generally  finger-like  in  shape  and  of  large  size. 

Three  forms  of  motility  may  be  distinguished: 
active  progressive  motion,  the  extrusion  of  pseudo- 
podia  without  progression,  and  movements  of  the 
cytoplasm. 

The  character  of  the  progressive  motion  in  freshly 
passed  feces  varies  considerably,  but  it  is  generally 
rapid  when  compared  to  the  sluggish  motion  of  Enta- 
moeba coli.  As  the  feces  become  cool  progressive 
motion  is  gradually  lost  and,  in  such  specimens,  may 
be  entirely  absent  or  so  sluggish  as  to  require  careful 
and  prolonged  observation  to  distinguish  it.  The 
organism  advances  by  throwing  out  clear  pseudopodia 
into  which  the  endoplasm  flows.  The  shape  of  the 
pseudopodia  varies  from  a  round,  broad  mass  of 
ectoplasm  to  long  slender  processes  with  rounded 
extremities.  Pointed  pseudopodia  so  frequently  ob- 
served in  free-living  species  of  amoeba*  are  never 
observed  in  any  of  the  parasitic  species  of  man.  The 
long,  slender  pseudopodia  are  most  frequently  ob- 
served in  the  rapidly  moving  organisms,  the  more 
rounded  pseudopodia  occurring  in  amrebae  possessing 
sluggish  motion. 

The  pseudopodia  are  always  hyaline  in  appearance 
when  first  extruded,  but  the  endoplasm  quickly  flows 


130  PARASITIC  AMCEB^E  OF  MAN. 

into  them,  giving  them  a  granular  appearance,  and 
in  rare  instances  amcebse  of  this  species  are  observed 
in  which  the  motion  is  so  rapid  that  the  distinction 
between  ectoplasm  and  endoplasm  cannot  be  made. 
In  Entamoeba  coli  it  is  often  very  difficult  to  dis- 
tinguish these  two  portions  of  the  cytoplasm  even 
when  the  organism  is  in  motion. 

In  Entamoeba  histolytica  the  flowing  of  the  endo- 
plasm into  the  pseudopodia  generally  occurs  very 
rapidly  and  it  often  appears  as  though  the  periphery 
of  the  endoplasm  ruptured,  allowing  the  contents  to 
rush  through  into  the  pseudopodia.  Not  infrequently 
a  constriction  is  present  in  the  pseudopodia  near  the 
boundary  of  the  endoplasm,  and  when  this  is  the  case, 
the  contents  may  be  seen  to  pass  slowly  through  the 
constriction,  the  nucleus,  vacuoles  and  red  corpuscles 
being  compressed  as  they  pass  through  the  narrow 
portion. 

When  motility  is  pronounced  the  amoebse  gener- 
ally progress  in  a  definite  direction  or  across  a  micro- 
scopic field  without  cessation  of  progressive  motion; 
in  other  instances  motion  will  occur  for  a  short  dis- 
tance in  one  direction,  followed  quickly  by  progres- 
sion in  another,  so  that  it  may  be  a  long  time  before 
the  amoeba  will  pass  out  of  the  field  of  a  one-sixth-inch 
objective. 

The  second  form  of  motility  is  frequently  ob- 


AMOEB.E  OF  THE  INTESTINAL  TRACT.     131 

served  in  organisms  which  have  been  exposed  to  room 
temperature  for  some  time,  and  consists  in  the  active 
extrusion  of  pseudopodia  unaccompanied  by  pro- 
gressive motion,  the  processes  of  ectoplasm  being  con- 
tinually projected  from  the  periphery  of  the  parasite 
and  as  quickly  withdrawn.  In  such  instances  the 
endoplasm  does  not  flow  into  the  pseudopodia,  as  a 
rule,  but  occasionally  this  occurs,  though  progressive 
movement  is  prevented  by  the  projection  of  new 
pseudopodia.  This  form  of  motility  is  only  observed 
in  organisms  which  have  been  exposed  to  unfavorable 
conditions,  as  lower  temperature  and  solutions  of 
chemical  substances,  so  that  it  is  fair  to  assume  that  it 
is  an  evidence  of  abnormal  environment. 

The  third  variety  of  motion  is  rarely  observed. 
It  may  be  called  an  intra-protoplasmic  form  of 
motility,  consisting  of  currents  evidently  produced 
within  the  endoplasm.  The  contents  of  the  endoplasm 
are  observed  to  be  in  motion  in  a  circular  manner, 
the  nucleus,  vacuoles,  red  blood  cells,  and  other  sub- 
stances being  whirled  about  within  a  boundary  formed 
by  the  ectoplasm,  while  an  undulatory  motion  of 
the  latter  is  sometimes  observed.  The  movement  of 
the  current  is  generally  slow,  but  it  may  be  very 
rapid,  and  may  continue  for  several  minutes. 

Of  the  significance  of  this  form  of  motion  we  are 
ignorant.  It  is  somewhat  similar  to  that  occurring 


PARASITIC  AMOEBJE  OF  MAN. 

in  Balantidium  coli  just  prior  to  encystment,  and 
also  to  the  whirling  motion  of  Paramceba  hominis,  a 
species  of  amoebae  parasitic  in  man,  in  which  encyst- 
ment is  always  preceded  by  this  form  of  motility. 
However,  in  the  case  of  Entamoeba  Jdstolytica  I  have 
never  observed  any  evidence  of  encystment,  although 
such  organisms  have  been  watched  for  hours.  In 
addition,  progressive  motion  may  be  resumed  after 
this  form  of  motility  has  occurred,  thus  indicating  that 
it  has  nothing  to  do  with  encystment. 

STAINED  PREPARATIONS. — This  species  of  amoebae 
may  be  stained  with  any  of  the  methods  which  have 
been  described,  the  preparations  having  been  either 
wet-  or  dry-fixed.  The  best  results  are  obtained  with 
Wright's  stain,  using  wet-fixed  preparations  or  with 
iron  hsematoxylin  preparations  which  have  been  fixed 
while  wet.  It  is  always  difficult  to  stain  these  para- 
sites and  many  preparations  will  have  to  be  examined 
before  one  can  expect  to  obtain  material  for  a  careful 
morphological  study.  I  have  found  that  Wright's 
stain  gives  good  results  and  that  with  it  one  is  able 
to  follow  the  changes  that  occur  in  the  nucleus  during 
reproduction. 

It  is  also  possible  with  this  stain  to  differentiate 
this  species  from  Entamoeba  coli,  the  larger  parasites 
being  differentiated  by  the  deep  blue  staining  of  the 
ectoplasm  and  the  dim  staining  of  the  endoplasm,  the 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     133 

opposite  being  true  of  Entamceba  coll.  In  the  smaller 
amoebae  this  distinction  cannot  be  easily  made,  but 
every  specimen  of  feces  containing  this  species  will 
show  some  organisms  in  which  the  distinctive  staining 
of  the  ectoplasm  and  endoplasm  will  be  found. 

The  nucleus  of  this  species  stains  very  poorly  as 
compared  with  that  of  Entamceba  coli,  on  account  of 
the  small  amount  of  chromatin  which  it  contains,  as 
well  as  from  the  fact  that  in  many  of  the  amoebae  the 
nucleus  is  undergoing  division  prior  to  sporulation. 
With  Wright's  stain  the  nuclear  chromatin  stains  a 
pale  red  or  pink,  while  if  iron  hsematoxylin  be  used 
for  staining  the  nucleus  is  dark  blue  or  almost  black 
in  color.  As  a  rule,  the  nucleus  appears  larger  in 
Wright-  or  Giemsa-stained  preparations  and  the 
chromatin  granules  appear  more  irregular  and 
massive. 

METHODS  OF  REPRODUCTION. — We  owe  our  first 
accurate  description  of  the  methods  of  reproduction 
of  Entamceba  histolytica  to  Schaudinn,  and  upon  the 
difference  in  these  methods  from  those  of  Entamceba 
coll  he  very  largely  based  his  classification  of  these 
two  species.  The  difference  in  the  methods  of  repro- 
duction of  Entamceba  histolytica  and  Entamceba  coli, 
amply  suffices  to  establish  the  two  species,  even  though 
we  had  no  other  evidence  of  their  specific  nature. 

Entamceba  histolytica  reproduces  by  simple  divi- 
sion by  budding  or  gemmation,  and  by  sporulation. 


134  PARASITIC  AMCEBJE  OF  MAN. 

Simple  division  occurs  as  in  Entamceba  coU,  the 
nucleus  dividing  into  two  almost  equal  portions  fol- 
lowed by  the  division  of  the  cytoplasm,  which  results 
in  the  production  of  two  motile  amoebae.  Schaudinn 
described  the  division  of  the  nucleus  as  amitotic  in 
character,  but  if  the  iron-hsemotoxylin  method  of 
staining  be  employed,  the  division  is  seen  to  be  mitotic, 
the  karyosome  dividing  with  the  formation  of  a  nu- 
clear spindle  and  a  central  spindle  formed  by  the 
division  of  the  centriola.  The  observations  concern- 
ing the  mitotic  division  of  the  nucleus  in  this  species 
have  been  confirmed  by  H.  Werner,  who  describes 
the  process  very  accurately. 

Reproduction  by  budding  or  gemmation  consists 
in  the  formation  of  small  daughter  amoebae  which  are 
pinched  or  budded  off  from  the  periphery  of  the 
mother  organism.  This  process  is  initiated  by  the 
distribution  of  the  nuclear  chromatin  to  the  cyto- 
plasm, a  portion  of  the  nucleus  undergoing  degenera- 
tion. The  chromatin  collects  into  small  meshes  form- 
ing a  portion  of  the  nucleus  of  the  young  amoebaa,  and 
is  finally  extruded  from  the  parasite,  together  with 
a  portion  of  the  cytoplasm.  This  process  is  quite 
similar  to  sporulation,  but  does  not  result  in  the 
formation  of  resistant  spores.  In  specimens  stained 
with  iron  hasmatoxylin  the  chromatin  appears  in  the 
form  of  dots  and  threads  distributed  in  the  cytoplasm 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     135 

but  with  the  Wright  or  Giemsa  stain  it  appears  in 
irregular  masses. 

Reproduction  by  sporulation  only  occurs  after 
vegetative  reproduction  has  taken  place  for  many 
generations.  Until  the  researches  of  Schaudinn  this 
method  of  reproduction  had  not  been  observed  in  any 
amoebae,  but  since  his  researches  were  published  it  has 
been  confirmed  by  numerous  investigators  and  serves 
to  distinguish  this  species  from  the  other  intestinal 
amoebse.  In  view  of  the  interest  attaching  to  Schau- 
dinn's  work  I  shall  quote  in  full  his  description  of 
this  method  of  reproduction. 

"  As  in  many  other  parasitic  protozoa  the  forma- 
tion of  these  stages  in  Entamoeba  histolytica  occurs 
only  after  a  lengthy  period  of  lively  increase,  when 
the  conditions  of  life  have  deteriorated.  In  dysentery 
this  is  simultaneous  with  the  commencement  of  heal- 
ing. Permanent  forms  ensue  when  the  feces  become 
firmer — or  in  more  correct  language,  healing  begins 
when  the  vegetative  increase  of  the  amoebae  ceases. 
During  the  height  of  the  disease  I  have  never  found 
these  permanent  stages. 

"  The  beginning  of  spore  formation  is  first  noticed 
in  the  nuclear  apparatus.  The  peripheral  chromatin 
zone  of  the  nucleus  broadens  and  extends  into  the 
nuclear  plasm,  the  nucleus  at  the  same  time  becoming 


136  PARASITIC  AMOEBAE  OF  MAN. 

less  differentiated  and  surrendering  large  quantities 
of  chromatin  to  the  cytoplasm.  In  the  stained  speci- 
mens the  casting  off  of  these  chromatin  granules  can 
be  followed  step  by  step.  The  amount  of  chromatin 
in  the  cytoplasm  increases  until  the  entire  organism 
appears  to  be  filled  with  it  while  the  remainder  of  the 
nucleus  degenerates. 

"  In  observing  these  forms  during  life  the  fol- 
lowing phenomena  are  noted:  The  nucleus  is  located 
at  the  periphery  of  the  parasite  generally  in  the  shape 
of  a  flat  disc  at  the  border  of  the  ectoplasm.  Some- 
times it  is  entirely  expelled  while  under  the  eye  of  the 
observer.  The  peripheral  ectoplasm  portion  appears 
at  first  entirely  homogeneous,  but  as  the  process 
proceeds  a  fine  fibrous  structure  is  observed  parallel 
to  the  surface,  indicating  the  formation  of  the  buds 
which  finally  project  from  the  surface  of  the  organ- 
ism. Gradually  these  small  elevations  multiply,  rise 
higher  upon  the  surface,  and  finally  separate  in  the 
shape  of  small  globules  measuring  from  3  to  7  microns 
in  diameter.  In  a  short  time  these  globules  without 
changing  in  structure  will  develop  upon  their  surface 
a  colorless  double  outline  membrane  which  in  a  few 
hours  becomes  brownish  in  color,  the  interior  of  the 
globule  appearing  structureless,  while  the  parent- 
amoeba  gradually  breaks  up  and  disappears. 

"  A  staining  of  this  series  of  stages  gives  the 


AMCEBJS  OF  THE  INTESTINAL  TRACT.     137 

following  results.  The  nucleus  gives  off  chromidia 
to  the  cytoplasm,  which  appear  to  multiply  and  scat- 
ter through  the  entire  organism,  the  nucleus  degen- 
erating and  either  entirely  dissolving  or  being  ex- 
truded. The  chromidia  withdraw  from  the  endoplasm 
and  collect  in  the  dense  fibrous  tissue  of  the  ecto- 
plasm, finally  permeating  the  latter  as  a  uniform 
reticular  chromidial  mass.  Ectoplasma  buds  filled 
with  a  chromidial  mass  then  protrude  upon  the  sur- 
face of  the  parasite,  and  are  finally  budded  off  from 
the  parent  body.  As  soon  as  the  sheath  of  these 
globules  is  formed  staining  substances  no  longer  act 
well. 

"If  the  yellowish  brown  sheath  now  forms,  even 
the  stained  preparations  will  no  longer  give  informa- 
tion concerning  the  structure  of  the  interior  of  the 
globules,  and  section  technique  likewise  fails,  for  the 
small  globules  do  not  yield  to  the  knife.  Hence  I 
am  unable  to  state  anything  in  regard  to  the  nuclear 
changes  occurring  in  the  interior  of  these  spores." 

Prior  to  the  observations  of  Schaudinn  regarding 
this  method  of  reproduction,  I  published  a  description 
of  Entamceba  histolytica  in  which  I  mentioned  the 
occurrence  of  bodies  within  the  cytoplasm  which  I 
thought  might  be  spores.  In  concluding  this  paper  I 
said: 


138  PARASITIC  AMCEB.E  OF  MAN. 

"  There  occur  in  all  but  the  degenerative  forms 
of  the  amoebae  small  round  or  oval,  dimly  stained 
areas,  uniform  in  appearance,  and  most  numerous  in 
the  large  full-grown  forms,  being  entirely  absent  in 
the  vacuolated  shells  of  amoeba?.  These  areas  re- 
semble similar  areas  in  stained  segmenting  malarial 
plasmodia,  and  which  are  in  them  due  to  the  young 
spores.  Reasoning  from  analogy  it  may  be  that  these 
areas  in  the  amoebae  are  also  spores." 

After  the  appearance  of  Schaudinn's  work  it  was 
very  apparent  that  the  bodies  which  I  described  were 
in  reality  the  small  masses  of  chromatin  which  form 
a  portion  of  the  nucleus  of  the  young  spores. 

I  have  been  able  to  confirm  Schaudinn's  descrip- 
tion of  this  method  of  reproduction  and  the  following 
description  gives  the  results  of  my  studies  of  this 
process  as  observed  in  living  and  stained  preparations 
of  Entamceba  histolytica,  using  both  wet  and  dry 
fixed  specimens. 

It  is  not  unusual  to  observe  in  the  f  eces  amoebae  in 
which  the  endoplasm  contains  refractile  granules  and 
rods  collected  in  irregular  masses  or  distributed 
throughout  the  organism.  These  bodies  are  the 
chromidia  which  have  been  liberated  by  the  breaking 
up  of  the  nucleus.  Werner  considers  that  part  of  the 
chromidia  is  derived  from  the  peripheral  nuclear 
chromatin  and  part  of  it  is  produced  by  the  karyo- 


;») 


#P9 


G 


FIG.  XVIII. —  Reproduction  by  budding  in  Entamceba  histolytica. 
(Craig.)  A,  organisms  showing  rods  and  granules  of  chromatin  in  the 
nucleus,  a  vacuole  containing  some  stained  substance  and  the  deeply  stained 
ectoplasm;  B,  illustrating  the  passing  of  the  nuclear  chromatin  into  the 
cytoplasm,  where  it  becomes  distributed  as  chromidia,  shown  in  C;  D,  the 
formation  of  secondary  nuclei  by  aggregations  of  chromidia;  E,  the  forma- 
tion of  spores  by  budding;  F,  spores  of  Entamceba  histolytica  after  libera- 
tion from  the  parent  organism. 


FIG.  XIX. — A   section   of    the   intestine  showing    Entamceba   histolytica 
within  the  mucous  membrane.      X  7.50. 


'AMCEEM  OF  THE  INTESTINAL  TRACT.     139 

some.  The  chromidia  appear  as  brightly  refractile 
granules  and  delicate  threads  with  enlarged  ends. 
Other  organisms  are  observed  in  which  the  chromidia 
are  collected  in  masses  near  or  within  the  ectoplasm, 
sometimes  distending  it.  This  appearance  is  caused 
by  the  grouping  of  the  chromidia  prior  to  the  libera- 
tion of  the  spores.  Still  other  organisms  are  observed 
in  which  refractile  masses  of  chromatin,  surrounded 
by  a  small  portion  of  cytoplasm,  are  in  a  process  of 
separation  from  the  parent  organism. 

The  spores  in  the  fresh  specimens  of  feces  appear 
as  round  or  oval  bodies  having  a  yellowish  refractile 
membrane  and  homogeneous  contents.  They  measure 
from  3  to  7  microns  in  diameter  and  on  account  of 
their  color  they  resemble  red  blood  corpuscles,  but 
may  be  differentiated  from  them  because  of  the  sur- 
rounding membrane. 

If  it  is  desired  to  follow  the  method  of  reproduc- 
tion in  the  stained  preparations  a  large  number  of 
specimens  must  be  examined  and  Wright's  stain  or 
better,  iron  hsematoxylin,  used  for  this  purpose.  The 
larger  amoebae  should  be  selected  for  study  and  the 
following  forms  illustrating  various  stages  in  the 
process  will  be  observed:  , 

1.  Amoebae  in  which  the  chromatin  of  the  nucleus 
stains  a  light  pink  and  is  arranged  in  the  form  of 
delicate  rods  and  granules. 


140  PARASITIC  AMGEB.E  OF  MAN. 

2.  Amoebag  in  which  the  chromatin  is  situated 
partly  within  the  nucleus  and  partly  near  it,  in  the 
endoplasm.    The  chromatin  appears  to  have  increased 
in  amount  and  stains  more  intensely.    This  form  illus- 
trates   the    beginning    of    the    distribution    of    the 
chromidia  to  the  endoplasm  and  the  degeneration  of 
the  nucleus. 

3.  Amreba?  in  which  the  chromatin  is  distributed 
in  very  faintly  stained  grains  and  rods  throughout 
the  endoplasm,  the  remainder  of  the  nucleus  having 
disappeared. 

4.  Amosbse  in  which  the  chromatin  is  collected 
into  small  clumps  situated  near  or  in  the  ectoplasm. 
These   organisms  represent   the   stage   just   before 
sporulation. 

5.  Amrebse    in    which    some    of    the    masses    of 
chromatin  are  arranged  within  the  ectoplasm  causing 
it  to  project  slightly,  and  in  which  one  or  more  of 
the   clumps   of   chromatin,    surrounded   by   a   little 
protoplasm,  is  partially  or  entirely  separated  from 
the  parent  organism.    In  some  of  these  an  unstained 
area  will  be  observed  surrounding  the  spore,  indicat- 
ing the  beginning  of  the  formation  of  the  sheath. 

From  the  description  of  the  various  forms  which 
may  be  observed  in  stained  preparations  it  is  evident 
that  the  entire  process  of  reproduction  described  by 
Schaudinn  can  be  followed  and  the  observations  of 
numerous  investigators  have  confirmed  his  results. 


AMOEBAE  OF  THE  INTESTINAL  TRACT. 

This  method  of  reproduction  definitely  differen- 
tiated this  species  of  amoeba  from  Entamaeba  coli,  in 
which  reproduction  occurs  in  an  entirety  different 
manner.  If  Schaudinn's  classification  rested  entirely 
upon  the  marked  differences  in  the  reproductive  cycle 
of  these  two  organisms  it  would  be  unassailable,  but 
in  addition  we  have  marked  differences  in  mor- 
phology, and  the  experimental  evidence  of  the  effect 
of  the  two  species  upon  susceptible  animals. 

Conjugation. — I  have  several  times  observed  a 
process  which  may  be  interpreted  as  conjugation  in 
this  species  of  amoeba.  Two  organisms  may  some- 
times be  noted  lying  in  contact,  while  marked  stream- 
ing of  the  protoplasm  of  each  is  present.  It  often 
appears  as  though  there  was  an  interchange  of  proto- 
plasm and  I  am  sure  that  at  times  I  have  seen  the 
nucleus  of  one  within  the  cytoplasm  of  the  other. 
Beside  the  streaming  motion  of  the  protoplasm  the 
organisms  are  frequently  observed  to  apparently  re- 
volve about  one  another,  while  still  attached,  this 
movement  alternating  with  the  motion  of  the  proto- 
plasm. 

Conjugation  has  also  been  described  in  this  species 
by  Werner,  and  he  states  that  one  of  the  organisms 
appears  to  differ  from  the  other,  being  clearer  and 
more  homogeneous  in  structure,  but  I  have  not  been 
able  to  notice  any  difference  in  the  appearance  of  the 
conjugants. 


PARASITIC  AMGEBJE  OF  MAN. 

It  is  impossible  at  present  to  be  sure  of  the  exact 
nature  of  this  process,  but  it  appears  to  me  more 
than  probable  that  it  is  an  instance  of  true  conjuga- 
tion. Such  a  process  has  been  described  as  occurring 
in  Ealantidium  coli  and  I  have  also  observed  it  in 
Entamceba  tetragena. 

RELATION  TO  DISEASE. — At  the  present  time 
almost  all  authorities  are  convinced  that  certain  forms 
of  dysentery  are  due  to  amoebae,  but  there  are  still  a 
few  students  of  the  subject  who  maintain  that  the 
amoebae  are  only  secondary  invaders.  In  support  of 
their  position  they  claim  that  these  organisms  are 
often  found  in  the  f eces  of  healthy  individuals  and  of 
those  suffering  from  other  diseases;  that  direct  in- 
fection with  amoebae  has  never  been  proved;  that  de- 
ductions based  on  experiments  upon  cats,  monkeys, 
and  other  animals  are  unsatisfactory,  because  such 
animals  suffer  naturally  from  dysentery;  that  the 
presence  of  amoebae  in  f  eces  is  a  natural  condition,  such 
organisms  being  normal  inhabitants  of  the  intestines ; 
and  finally  that  the  pathogenic  amoebae  have  never 
been  cultivated. 

These  arguments  can  all  be  satisfactorily  answered 
by  the  results  of  research  accomplished  during  the 
past  seven  years.  We  now  know  that  the  presence 
of  amoebae  in  healthy  individuals  and  in  those  suffer- 
ing from  diseases  other  than  dysentery  is  explained 


AMCEBJE  OF  THE  INTESTINAL  TRACT. 

by  the  existence  of  a  non-pathogenic  species,  Enta- 
mceba  coll;  that  experiments  upon  animals  are  re- 
liable provided  they  are  properly  controlled;  that 
direct  infection  of  susceptible  animals  has  been 
abundantly  proven;  and  that  the  argument  that  the 
pathogenic  species  have  not  been  cultivated  is  not  a 
valid  one  as  disproving  their  relation  to  dysentery, 
as  many  other  organisms  undoubtedly  connected  with 
disease  have  not  been  cultivated. 

In  support  of  the  causative  relation  of  certain 
species  of  amoebae  to  dysentery  we  have  the  following 
facts: 

1.  The    absolutely    characteristic    pathology    of 
amoebic  dysentery. 

2.  The    constant    presence    of    the    pathogenic 
species  in  the  characteristic  lesions  and  their  absence 
from  the  lesions  of  other  kinds  of  dysentery. 

3.  The  constant  presence  of  the  amoebae  in  the 
peculiar  form  of  abscess  of  the  liver  complicating 
amoebic  dysentery. 

4.  The  experimental  production  by  feeding  and 
inoculation  experiments,  of  a  disease  in  susceptible 
animals  presenting  the  same  pathological  lesions  as 
those  of  human  amoebic  dysentery. 

The  evidence  mentioned  above  applies  in  the  case 
of  Entamceba  histolytica  and  Entamceba  tetragena, 
the  two  pathogenic  species  which  had  been  thoroughly 


144  PARASITIC  AMOEBJE  OF  MAN. 

studied,  and  this  evidence  will  now  be  considered  in 
detail. 

The  Pathology  of  Amoebic  Dysentery. — It  is 
not  my  intention  to  discuss  fully  the  pathology  of  the 
form  of  dysentery  due  to  Entamceba  histolytica,  but 
to  simply  call  attention  to  the  characteristic  lesions  of 
this  condition.  It  is  now  well  recognized  that  great 
epidemics  of  dysentery  occur  in  which  amoebae  cannot 
be  demonstrated  in  the  lesions  of  the  disease,  and  that 
such  epidemics  are  due  to  a  group  of  bacteria.  This 
type  of  the  disease  is  known  as  bacillary  dysentery, 
and  the  lesions  present  differ  markedly  from  those 
found  in  amoebic  dysentery.  In  the  bacillary  type 
the  characteristic  lesion  is  a  general  superficial  ulcera- 
tion  of  the  mucous  membrane  of  the  colon  accom- 
panied by  intense  congestion,  while  in  the  more 
chronic  forms  the  ulcers  may  penetrate  into  the  sub- 
mucosa  and  a  general  gangrenous  condition  of  the 
bowel  may  result,  but  to  one  who  has  had  experience 
in  autopsy  work  upon  this  form  of  dysentery  a  glance 
is  sufficient  to  distinguish  it  from  the  amoebic  type. 
The  lesions  of  the  latter  form  of  dysentery  cannot  be 
confused  with  those  found  in  any  other  variety, 
although  some  authorities  have  tried  to  prove  that  the 
lesions  of  amoebic  dysentery  do  not  differ  markedly 
from  those  of  the  bacillary  type.  Such  statements 
can  only  be  based  upon  a  very  limited  experience  for 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     145 

even  the  tyro  will  not  be  confused  if  he  has  seen  a 
few  cases  of  both  varieties  upon  the  autopsy  table. 
I  have  performed  several  hundred  autopsies  upon 
cases  of  dysentery,  including  both  the  bacillary  and 
amoebic  types,  and  can  state  positively  that  it  is  im- 
possible to  confuse  the  pathology  of  the  amoebic  type 
with  that  of  the  bacillary. 

The  lesions  of  amoebic  dysentery  are  most  com- 
monly observed  in  the  rectum  and  just  below  the  ileo- 
csecal  valve.  In  mild  cases  they  may  be  confined  to 
one  of  these  regions,  while  in  the  most  severe  cases 
the  entire  colon  may  be  invaded.  As  showing  the 
relative  frequency  of  the  lesions  in  various  portions 
of  the  large  intestine  I  may  say  that  out  of  78  cases 
no  less  than  57  showed  lesions  below  the  ileocaecal 
valve  and  in  the  rectum,  the  intervening  portion  of 
the  intestine  being  uninvolved;  twelve  cases  showed 
lesions  extending  the  entire  length  of  the  colon,  but 
invariably  most  severe  in  the  rectum  and  below  the 
ileocascal  valve;  while  the  remaining  nine  showed 
lesions  only  in  the  rectum  and  for  a  short  distance 
above  the  sigmoid  flexure.  In  only  two  of  the  cases 
that  came  to  autopsy  have  I  observed  any  extension 
of  the  disease  above  the  ileocsecal  valve. 

The  most  characteristic  lesions  of  the  early  stage 
of  amoebic  infection  are  small  nodular  areas  which 
project  from  the  summit  of  the  folds  of  the  mucous 

10 


146  PARASITIC  AMCEB.E  OF  MAN. 

membrane  into  the  lumen  of  the  intestine.  The 
mucous  membrane  covering  them  is  generally  in- 
flamed, and  when  incised  they  are  found  to  contain  a 
yellowish  or  a  greenish-yellow  viscid  fluid  of  a 
gelatinous  consistence,  which,  upon  microscopic  ex- 
amination, is  seen  to  be  composed  of  degenerated  cel- 
lular material,  mucus,  and  actively  motile  amoebae. 
These  nodular  elevations  mark  the  situation  of  the 
ulceration  which  develops  later. 

The  next  stage  of  amoebic  dysentery  is  indicated 
by  the  appearance  of  small  ulcers  formed  by  the 
necrosis  of  the  mucous  membrane  covering  the  nodu- 
lar elevations  just  mentioned.  The  base  of  these 
ulcers  is  surrounded  by  inflamed  mucous  membrane, 
while  the  edges  are  ragged  in  appearance,  and  the 
floor,  situated  in  the  submucous  coat  of  the  intestine, 
is  covered  with  the  gelatinous  material  which  has  been 
described.  The  ulcers  spread  by  invasion  of  the  sur- 
rounding structure  both  laterally  and  downward,  be- 
coming larger  and  larger,  finally  reaching  the  muscu- 
lar coat  of  the  intestine.  In  some  instances  the  small 
ulcers  are  pretty  generally  distributed  and  all  of 
about  the  same  size,  but  in  a  majority  of  cases  both 
the  small  and  large  ulcers  occur  together,  and  in 
many  cases  one  may  trace  the  entire  pathology  of  the 
disease,  from  the  initial  lesion,  represented  by  the 
intact  nodular  mass,  to  the  large  ulcer  involving  all  of 
the  coats  of  the  intestine. 


AMGEBJE  OF  THE  INTESTINAL  TRACT.     147 

The  invasion  of  the  mucous  membrane  laterally 
leads  to  the  formation  of  sinuses  beneath  this  mem- 
brane, connecting  the  neighboring  ulcers.  This  lesion 
is  one  most  characteristic  of  amoebic  dysentery  and 
does  not  occur  in  any  other  form.  Upon  opening 
such  sinuses  it  is  generally  found  that  they  contain 
gelatinous  material  similar  to  that  contained  within 
the  nodules  and  microscopic  examination  reveals  the 
presence  of  numerous  amoeba?. 

In  aggravated  cases  the  necrosis  of  the  mucous 
and  submucous  coat  of  the  intestine  as  well  as  the 
coalescence  of  smaller  ulcers,  leads  to  the  formation  of 
irregular  ulcers  of  large  extent,  the  mucous  mem- 
brane between  them  presenting  deep,  irregular  chan- 
nels produced  by  the  necrosis  of  the  tissue  covering 
the  sinuses,  while  the  whole  surface  of  the  intestine  is 
covered  with  partly  detached  shreds  of  necrotic  mem- 
brane. This  so-called  "  buffalo  skin  "  appearance  is 
very  frequently  present  in  severe  cases  of  amoebic 
dysentery  and  is  very  characteristic  of  that  disease. 

It  is  very  difficult  to  describe  the  various  forms 
of  ulceration  which  may  be  present  in  the  intestine 
in  amoebic  infection.  A  typical  ulcer  may  be  said  to 
have  the  following  structure:  the  edges  are  consider- 
ably raised  from  the  surface  of  the  mucous  membrane 
and  are  much  undermined,  presenting  a  very  charac- 
teristic shaggy  appearance  due  to  necrotic  tissue ;  the 


148  PARASITIC  AMCEB^  OF  MAN, 

floor  of  the  ulcer  may  be  rough  or  smooth,  the  older 
ulcers  having  a  smooth  floor  while  in  the  more  recent 
ones  the  floor  is  covered  with  necrotic  material,  pus, 
and  blood.  In  the  majority  of  the  ulcers  the  floor 
is  formed  by  the  submucous  coat  of  the  intestine,  but 
in  all  advanced  cases  ulcers  will  be  observed  in  which 
the  floor  is  formed  by  the  muscular  coat  of  the  intes- 
tine. The  smaller  ulcers  are  generally  round  or  oval 
in  shape,  but  the  larger  ones  are  more  irregular  in 
shape.  The  ulcers  vary  in  size  from  0.5  cm.  to  from 
8  to  10  cm.  in  diameter.  Frequently  the  entire 
mucous  membrane  of  a  considerable  portion  of  the 
intestine  has  been  destroyed  by  the  coalescence  of  two 
or  more  large  ulcers,  and  I  have  repeatedly  observed 
ulcers  encircling  the  bowel  and  measuring  as  much 
as  6  to  8  cm.  in  the  short  diameter. 

The  mucous  membrane  between  the  ulcerations  in 
mild  infections  may  appear  normal,  but  in  the  more 
severe  types  it  is  always  inflamed,  thickened,  and 
covered  with  mucus  and  pus.  The  muscular  coat  of 
the  intestine  appears  to  offer  a  barrier  to  the  exten- 
sion of  the  disease,  but  this  coat  may  be  destroyed  and 
perforation  of  the  intestine  may  occur.  In  chronic 
cases  the  muscular  and  submucous  coats  of  the  in- 
testine become  greatly  thickened  and  sometimes 
almost  cartilaginous  in  consistence. 

The  exact  manner  in  which  the  amoebae  produce 


lyto"!!Sf\vs5^pS 

'^^;!v%>r^^   -'-r^^\V^,       V,       '."•1&./W; 

lkiv'^ 


XXI 


FIGS.  XX  and  XXI. —  Sections  of  the  intestine  showing  numerous  amoebae 
within  the  tissues.     (After  Councilman  and  Lafleur.) 


AMOEBAE  OF  THE  INTESTINAL  TRACT.     149 

lesions  of  the  intestine  is  not  known.  That  they  are 
able  to  penetrate  the  epithelial  lining  of  this  organ  has 
been  abundantly  proven  by  the  work  of  Jiirgens, 
Schaudinn,  and  others,  and  by  their  presence  in  the 
tissues  as  shown  in  microscopic  sections.  It  is  possible 
that  they  secrete  a  toxin  which  causes  the  initial  de- 
struction of  the  epithelial  cells  which  enables  them  to 
penetrate  beneath  the  mucous  membrane,  and  that 
this  toxin  has  much  to  do  with  the  production  of  the 
lesions  in  the  deeper  tissues,  but  of  this  we  have  no 
definite  proof. 

In  microscopic  sections  of  the  intestine  of  patients 
dying  of  amoebic  dysentery,  it  may  be  stated  that, 
almost  without  exception,  the  amoebae  are  present  in 
the  necrotic  tissue  forming  the  wall  of  the  ulcers  and 
in  the  surrounding  tissue.  They  are  most  abundant 
in  the  submucous  coat,  but  are  often  observed  in  the 
muscular  coat  when  the  ulceration  has  extended  to 
that  region.-  When  present  they  are  always  sur- 
rounded by  numerous  leucocytes  and  other  evidence 
of  inflammatory  action.  They  are  most  numerous 
in  the  areas  which  are  situated  nearest  the  region  of 
greatest  degenerative  changes,  and  it  is  rather  rare 
to  find  them  in  the  necrotic  tissue  overlying  the  ulcers 
or  composing  the  edges.  Amoebae  may  be  observed 
lying  within  the  lymph  spaces  and  sometimes  within 
the  lymphatics  and  veins.  When  the  disease  has  ex- 


150  PARASITIC  AMCEB^E  OF  MAN. 

tended  to  the  muscular  coat  the  amoebae  are  found 
lying  in  the  intermuscular  septa  and  in  this  situation 
they  may  be  observed  arranged  in  rows  and,  where 
the  tissue  is  not  compact,  scattered  in  irregular 
groups.  In  whatever  region  they  are  present,  how- 
ever, there  is  always  evidence  of  an  inflammatory 
process. 

It  is  difficult  to  say  how  much  of  the  pathologic 
process  present  is  actually  due  to  the  amoebae,  for 
after  the  formation  of  ulcers  we  must  remember  that 
we  are  dealing  with  a  mixed  infection.  Multitudes 
of  bacteria  are  present  which  may  have  much  to  do 
with  the  morphology  of  the  lesions,  but  the  fact  re- 
mains that  these  lesions  are  not  the  same  in  appear- 
ance as  those  produced  by  bacteria  alone,  thus  indicat- 
ing that  the  characteristic  picture  of  amoebic  infection 
is  in  all  probability  due  to  the  specific  action  of  the 
amoebae. 

There  are  three  lesions  present  in  this  disease 
which  are  absolutely  diagnostic: 

1.  The  nodular  thickenings  situated  in  the  mucous 
membrane  which,  when  incised,  are  found  to  be  filled 
with  gelatinous  material. 

2.  Ulcers  having  greatly  thickened  walls  raised 
from  the  surrounding  mucous  membrane  and  present- 
ing shaggy,  yellowish-brown  edges,  which  are  always 
undermined.      These   ulcers   are   often   covered   by 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     151 

necrotic  membrane  which  upon  removal  reveals  the 
interior  of  the  ulcer  filled  with  mucus,  pus  or  blood, 
the  floor  being  formed  by  the  submucous  or  muscu- 
lar coat.  The  presence  in  advanced  cases  of  numer- 
ous ulcers  causes  the  interior  of  the  intestine  to  re- 
semble the  rough,  yellowish-brown,  shaggy  appear- 
ance presented  by  old  buffalo  skins. 

3.  The  almost  invariable  presence  of  irregular 
sinuses  connecting  the  ulcers  and  situated  beneath  the 
mucous  membrane. 

Abscess  of  the  Liver. — A  considerable  proportion 
of  patients  suffering  from  amoebic  dysentery  develop 
abscess  of  the  liver,  and  it  is  now  a  well  established 
fact  that  the  pathogenic  amoeba?  cause  this  peculiar 
form  of  liver  abscess. 

The  frequency  of  this  complication  is  variously 
stated  by  different  observers,  according  as  the  per- 
centage is  based  upon  autopsy  reports  or  upon  the 
total  number  of  cases  observed.  Kartulis  found  that 
55  per  cent,  of  five  hundred  cases  coming  to  autopsy 
showed  amoebic  abscess  of  the  liver;  Zancarol  found 
that  amoebic  abscess  of  the  liver  occurred  in  59  per 
cent,  of  444  cases  that  came  to  autopsy;  Smith  in 
45  autopsies  upon  amoebic  dysentery  cases  found  this 
complication  in  84.4  per  cent. 

Councilman  and  Lafleur  collected  the  data  upon 
1429  cases  of  amoebic  dysentery,  of  which  only  21 


152  PARASITIC  AMGEBJE  OF  MAN. 

per  cent,  were  complicated  by  abscess  of  the  liver. 
In  745  cases  of  amoebic  dysentery  which  I  observed, 
in  which  amoebse  were  demonstrated  in  the  feces,  ab- 
scesses occurred  in  only  about  5  per  cent.,  but  in  78 
fatal  cases  this  condition  was  observed  in  nearly 
33  per  cent.,  and  this  well  illustrates  the  difference 
in  statistics  obtained  by  considering  the  subject 
from  these  two  standpoints.  However,  I  must  admit 
that  the  percentage  is  much  lower  in  the  cases  I  have 
observed  than  is  usually  reported. 

The  number  of  abscesses  present  in  the  liver  varies 
from  one  to  20  or  more.  For  many  years  it  was 
considered  that  the  single  abscess  was  characteristic 
of  the  amoebic  type,  but  this  statement  is  not  borne 
out  by  autopsy  experience.  While  the  single  large 
abscess  is  often  observed  in  these  cases,  multiple  ab- 
scesses are  almost  as  frequently  observed,  and  in  my 
experience  over  50  per  cent,  of  fatal  cases  have  shown 
multiple  abscesses.  Sometimes  the  entire  liver  is  filled 
with  them,  but  in  such  instances  there  is  always  a 
mixed  infection  and  many  of  the  abscesses  are  due 
to  bacteria. 

The  amoebic  abscess  occurs  most  frequently  in  the 
right  lobe  of  the  liver,  the  favorite  location  being  at 
the  dome,  close  to  the  attachment  of  the  diaphragm, 
or  the  under  surface  near  the  hepatic  flexure  of  the 
colon.  Even  in  cases  where  multiple  abscesses  are 
present  the  largest  and  oldest  abscesses  are  almost 


AM(EBJG  OF  THE  INTESTINAL  TRACT.     153 


invariably  found  in  the  right  lobe.  I  have  observed 
but  three  cases  in  which  the  findings  at  autopsy  proved 
that  abscess  formation  was  confined  to  the  left  lobe. 
My  experience  in  this  respect  has  been  borne  out  by 
others,  especially  by  Rouis,  who  collected  639  cases 
of  abscess  of  the  liver  in  which  the  location  of  the 
abscesses  was  as  follows:  435  or  17.8  per  cent,  were 
situated  in  the  right  lobe;  85  or  13.3  per  cent,  were 
situated  in  the  left  lobe,  and  2  or  0.3  per  cent,  in  the 
lobus  Spigelii. 

The  following  table  illustrates  the  location  of  the 
abscesses  in  24  cases  which  I  have  observed  at  autopsy: 

TABLE  II. 

ILLUSTRATES  THE  NUMBER  AND  LOCATION  OF  ABSCESSES  IN  TWENTY-FOUR 
FATAL  INFECTIONS  WITH  ENTAMCEBA  HISTOLYTICA. 


I 

•o 

l_ 

1 

2 
3 
4 
5 
(> 
7 
8 

10 
11 

12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 

Abscesses. 

Single. 

Multiple. 

Number. 

Location. 

Ye's 
Yes 

Yes 

Yes 

Yes 

Yes 
Yes 

Yes 

Yes 
Yes 

Yes 
Yes 
Yes 
Yes 
Yes 

Yes 

Yes 
Yes 

Yes 

Yes 
Yes 

Yes 

Yes 
Yes 

6 
8 
4 
13 
8 
1 
1 
2 
1 
17 
Too  numerous 
to  count 
10 
1 
3 
5 
1 
3 
1 
1 
Very  numerous 

30 
4 

1 

Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 

Right  lobe 

Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe, 
Right  lobe 
Right  lobe 
Right  lobe 
Right  lobe 

5... 

Left  lobe  1 

8 

Left  lobe  0 

3  
8  .. 

.  Left  lobe  1 
Left  lobe  5 

g 

Left  lobe  0 

0  

Left  lobe  1 

0  . 

Left  lobe  1 

2  

.  Left  lobe  0 

Lobus  Spigelii. 
16 

Left  lobe  1 

In  both  lobes. 
2 

Left  lobe  8 
.  Left  lobe  0 
.  Left  lobe  0 
.  Left  lobe  0 
.  Left  lobe  1 
Left  lobe  0 

1  
3  
5  

0  
3 

1  

.  Left  lobe  0 
.  Left  lobe  0 

.  Left  lobe  0 
.  Left  lobe  7 
.  Left  lobe  0 
.  Left  lobe  0 

1  
all  but  2 

23  
4... 

154 


PARASITIC  AMOEBA  OF  MAN. 


The  rupture  of  a  liver  abscess  is  of  comparatively 
frequent  occurrence  and  may  take  place  into  the  ab- 
dominal cavity,  the  pleura,  the  pericardium,  or  into 
adjoining  viscera.  Of  24  cases  observed  at  the  Army 
General  Hospital,  in  San  Francisco,  no  less  than 
seven  ruptured  before  death,  five  into  the  right 
pleural  cavity,  and  two  into  the  left  pleural  cavity 
and  the  pericardium.  As  this  question  is  of  con- 
siderable surgical  importance  the  following  table  illus- 
trating the  place  of  rupture  in  over  100  cases  may 
prove  of  service: 

TABLE  III. 
ILLUSTRATING  THE  SITE  OF  RUPTURE  IN  AMCEBIC  ABSCESS  OF  THE  LIVER. 


S3 

, 

, 

g 

==*«• 

1 

| 

.2 

oj 

1 

Observers. 

o  c 

TO    X 

02    t-i 

| 

g 

. 

. 

1 

-O 

8 

£ 

1 

03 

J£ 

'C 

9 

g 

o 

| 

^J 

t 

33 

6 

& 

s 

3 

6 

OJ 

« 

M 

3 

Waring  

300 

68 

14 

28 

15 

2 

1 

1 

3 

2 

2 

Dutroulau  

66 

25 

2 

10 

7 

1 

1 

Rouis 

162 

54 

17 

1  4- 

* 

Haspel  

25 

6 

4 

2 

Cambay  

10 

3 

2 

Howard  

6 

5 

5 

Craig  

24 

7 

2 

When  very  large  or  situated  near  the  surface  an 
amoebic  abscess  is  often  visible  externally,  but  fre- 
quently they  are  only  discovered  upon  section  of  the 
liver;  when  multiple  abscesses  are  present  only  one 
or  two  may  be  visible,  the  others  being  situated  deep 
within  the  organ.  In  such  instances  a  careful  exam- 
ination will  always  disclose  the  fact  that  there  is 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     155 

always  one  which  shows  by  its  size  and  the  thickness 
of  the  abscess  wall  that  it  is  of  longer  duration  than 
the  others. 

The  contents  of  an  amoebic  abscess  vary  with  the 
character  of  the  infection.  In  those  abscesses  in  which 
the  amoebge  are  present  in  pure  culture,  the  material 
contained  within  them  is  characteristic,  consisting  of 
a  semi-fluid,  yellowish-red,  or  chocolate-colored  mass, 
containing  shreds  of  necrotic  tissue,  blood,  a  few  pus 
cells,  and  amoebae.  This  material  does  not  resemble 
pus  unless  there  is  a  mixed  infection  with  suppurative 
bacteria,  but  if  such  bacteria  be  present  the  contents 
of  the  liver  abscess  resemble  that  of  other  abscesses 
due  to  bacterial  agencies. 

The  character  of  the  abscess  wall  is  typical  in 
those  instances  in  which  the  amoebae  are  present  alone. 
Internally  it  is  covered  with  shreds  of  necrotic  tissue 
giving  it  a  peculiar  shaggy  appearance.  When  such 
an  abscess  is  washed  out  it  will  sometimes  be  found 
that  these  necrotic  shreds  reach  across  it,  and  I  have 
observed  cases  in  which  all  trace  of  liver  substance 
within  the  abscess  had  been  lost,  except  the  connective 
tissue  framework  of  the  organ  which,  being  more  re- 
sistent  to  necrosis  than  the  other  elements,  still  per- 
sisted as  shreds  of  tissue  crossing  the  abscess  cavity. 
This  typical  appearance  of  the  wall  is  observed  most 
frequently  in  medium  size  abscesses,  as  in  the  very 


156  PARASITIC  AMOEBA  OF  MAN. 

old  abscesses  a  mixed  infection  is  generally  present 
and  the  wall  of  the  cavity  is  generally  smooth  or 
moth-eaten  in  appearance.  In  the  smaller  abscesses 
the  interior  is  generally  almost  smooth  in  appearance, 
while  in  the  very  smallest  abscesses  no  distinct  wall 
can  be  differentiated. 

The  appearance  presented  by  sections  of  the  liver 
abscess  under  the  microscope  varies  with  the  age  of 
the  process.  In  the  earliest  stage,  there  is  simply  a 
collection  of  leucocytes  and  connective-tissue  cells, 
with  some  congestion  of  the  capillaries  in  the  vicinity, 
and  such  sections  stained  by  Mallory's  method  will 
sometimes  show  amoebae,  but  not  as  a  rule.  In  those 
abscesses  which  have  a  well-defined  wall  the  centre 
of  the  abscess  cavity  is  seen  to  consist  of  necrotic 
epithelium,  lymphoid  cells,  and  leucocytes,  together 
with  bacteria,  and  more  or  less  caseous  material. 
Newly  formed  bile  channels  are  often  observed,  and 
examination  of  the  periphery  of  the  small  abscesses 
shows  that  the  process  commences  in  the  interlobular 
areas.  The  wall  of  these  abscesses  is  formed  by 
connective  tissue  considerably  infiltrated  by  leuco- 
cytes and  young  connective-tissue  cells,  and  amoebge 
may  sometimes  be  demonstrated.  The  liver  cells  in 
the  immediate  vicinity  are  undergoing  necrosis  and 
the  bile  ducts  and  capillaries  are  congested,  the  bile 
ducts  often  being  obliterated  or  encroached  upon  by 


AM(EBJE  OF  THE  INTESTINAL  TRACT.     157 

the  rapidly  growing  connective  tissue.  In  the  large 
abscesses,  which  have  a  rather  thick  wall,  sections  show 
at  the  inner  border  of  the  abscesses  more  or  less 
necrotic  material,  while  externally  the  fibrous  tissue 
is  very  marked,  the  appearance  being  that  of  granu- 
lation tissue,  the  cells  of  which  are  mostly  uninuclear. 
More  externally  is  a  layer  of  less  dense  connective 
tissue  infiltrated  with  spindle  cells  and  small  round 
connective-tissue  cells.  This  infiltration  varies  with 
the  age  of  the  abscess.  When  the  fibrous  wall  is 
very  thick  the  cellular  infiltration  is  not  as  great  as 
where  the  fibrous  tissue  is  of  more  recent  formation. 

The  amoebae  are  found  in  the  abscess  wall  in  the 
zone  of  necrosis,  generally  near  the  border  of  the 
connective-tissue  portion  of  the  wall,  which  is  infil- 
trated by  small  round  cells.  From  this  it  will  be 
seen  that  amoebae  will  but  seldom  be  found  in  the 
very  old  abscesses  showing  little  necrosis  and  a  very 
thick  and  dense  fibrous  wall.  They  are  found  most 
commonly  in  the  medium-sized  abscesses  presenting 
evidences  of  marked  necrosis  of  the  liver  tissue. 

In  the  contents  of  liver  abscesses,  which  are  due 
wholly  to  the  amoebae,  are  found  shreds  of  necrotic 
tissue,  degenerative  liver  cells,  red  blood  corpuscles, 
granular  material,  and  amoebae.  It  is  remarkable 
how  rare  are  the  pus  corpuscles  which  are  found  in 
ordinary  pus,  sometimes  the  entire  field  of  the  micro- 


158  PARASITIC  AMCEBJE  OF  MAN. 

scope  showing  only  from  one-half  dozen  to  a  dozen. 
Amoebae  are  not  always  found  in  the  contents  of  the 
liver  abscesses,  especially  in  the  older  ones,  but  gen- 
erally scrapings  from  the  walls  will  demonstrate 
them.  In  the  abscesses  in  which  there  is  a  mixed 
infection,  pus  corpuscles  are  found  as  in  ordinary 
pus,  together  with  various  bacteria,  chiefly  micrococci. 
The  contents  of  the  liver  abscess  may  be  sterile 
except  for  the  amoebae,  or  there  may  be  a  mixed  in- 
fection with  various  bacteria.  In  the  smaller  abscesses 
it  is  generally  found  that  the  pus  is  sterile,  while  in 
the  large  there  is  generally  a  mixed  infection.  There 
has  been  considerable  discussion  as  to  the  relative 
importance  of  amoebae  and  bacteria  in  the  production 
of  these  liver  abscesses,  some  authorities  claiming  that 
the  condition  is  produced  entirely  by  the  amoebae, 
while  others  insist  that  it  is  due  to  a  mixed  infection. 
At  the  present  time,  however,  it  is  generally  conceded 
that  amoebae  are  capable  of  producing  in  the  liver  the 
characteristic  form  of  abscess  which  I  have  described, 
and  that  when  there  is  a  mixed  infection  the  abscesses 
lose  their  characteristic  features  and  resemble  those 
found  in  other  portions  of  the  body.  The  amoeba 
undoubtedly  produce  the  abscesses  primarily,  but  they 
afterward  become  infected  with  other  organisms, 
especially  streptococci,  staphylococci,  and  Bacillus 
coli  communis.  An  examination  of  the  contents  bac- 


AMQEB.E  OF  THE  INTESTINAL  TRACT.     159 

teriologically  has  resulted,  in  my  experience,  in  about 
40  per  cent,  of  the  cases  showing  a  mixed  infection 
with  some  other  organism.  My  observations  as  re- 
gards the  bacteriology  of  the  pus  in  liver  abscesses 
confirms  that  of  many  others  who  have  found  that  in 
about  one-half  the  cases  there  is  a  mixed  infection. 
It  is  in  these  cases,  also,  that  we  find  the  yellow  or 
greenish  pus,  rather  than  the  reddish  or  chocolate- 
colored  pus  which  is  so  typical  of  amoebic  infection. 
As  regards  the  frequency  with  which  other  organisms 
are  found,  Futcher  investigated  27  cases,  in  which  he 
found  Staphylococcus  aureus  in  6,  Bacillus  coli  com- 
munis  in  5,  usually  associated  with  other  organisms; 
Streptococcus  pyogenes  in  3,  and  Micrococcus 
lanceolatus  and  Bacillus  pyocyaneus  in  one,  each; 
while  12  showed  only  the  amoeba?.  Councilman  and 
Lafleur,  in  2  cases,  found  Bacillus  coli  communis  in 
one.  Kruse  and  Pasquale  found  streptococci  in  three 
dysenteric  abscesses,  staphylococci  in  two,  and  bacilli 
resembling  those  of  typhoid  (probably  coli  com- 
munis) in  four. 

The  peculiar  form  of  liver  abscess  which  I  have 
described  is  associated  only  with  certain  forms  of 
dysentery  due  to  infection  with  pathogenic  amoeba?. 
To  one  who  has  had  sufficient  experience  in  the  study 
of  such  forms  of  dysentery  there  can  be  no  doubt 
of  the  etiologic  relationship  of  the  amoeba?  to  it  and 


160  PARASITIC  AMCEB^E  OF  MAN. 

to  the  liver  abscesses  occurring  in  these  cases.  While 
liver  abscesses  occur  without  the  presence  of  amoebae, 
and  even  in  cases  of  dysentery  which  are  not  due  to 
amoebae,  this  is  no  argument  against  the  etiologic  im- 
portance of  the  parasite.  In  my  studies  of  the  form 
of  dysentery  which  is  due  to  infection  with  the  Shiga 
bacillus,  or  bacilli  belonging  to  that  group,  I  have 
observed  abscess  of  the  liver,  but  the  abscesses  were 
so  different,  both  in  macroscopic  and  microscopic 
pathology,  from  those  due  to  amoebae  that  no  mistake 
could  be  made  in  distinguishing  them. 

The  question  has  been  raised  as  to  whether  an 
amoebic  abscess  of  the  liver  ever  occurs  primarily, 
no  other  symptom  of  amoebic  infection  having  been 
noticed,  and  there  can  be  no  doubt  that  a  few 
authentic  instances  have  been  reported,  such  as  the 
case  recorded  by  Buxton  in  the  person  of  a  woman 
who  died  at  the  Philadelphia  Hospital.  The  autopsy 
showed  four  large  abscesses  in  the  right  lobe  and  one 
in  the  left  lobe  of  the  liver  the  pus  of  each  containing 
amoebae,  but  the  most  careful  examination  of  the 
intestine  showed  no  evidence  whatever  of  dysenteric 
infection.  Such  a  case  as  this,  recorded  by  a  com- 
petent observer,  is  conclusive  evidence  that  amoebic 
abscess  of  the  liver  may  occur  without  a  pre-existing 
dysentery.  In  the  vast  majority  of  cases,  however, 
there  is  a  history  of  dysentery  and  the  abscess  occurs 


AMGEB.E  OF  THE  INTESTINAL  TRACT.     161 

as  a  secondary  condition.  It  should  be  remembered 
in  this  connection  that  it  is  not  necessary  that  well 
marked  symptoms  of  dysentery  be  present  or  that 
the  disease  be  in  an  acute  stage,  for  many  cases  may 
have  entirely  recovered  from  the  dysenteric  symptoms 
before  the  abscess  of  the  liver  is  discovered  or  has 
developed. 

The  path  by  which  the  amoeba?  reach  the  liver 
from  the  intestine  undoubtedly  varies  in  different 
patients.  They  may  reach  the  liver  through  the  blood- 
vessels, the  lymphatics,  or  through  the  peritoneum. 
It  is  conceded  by  most  authorities  that  the  most 
probable  way  is  through  the  portal  vein,  and  in  sec- 
tions of  the  intestine  amoebae  are  frequently  observed 
lying  in  close  proximity  to  the  capillaries,  and  in 
several  instances  I  have  observed  them  actually  within 
the  capillaries.  Councilman  and  others  consider 
that  the  most  common  way  is  through  the  peritoneal 
cavity  and  amoebae  have  been  demonstrated  in  the 
exudate  in  peritonitis  and  in  the  exudate  covering 
the  surface  of  the  liver.  This  path  of  infection  may 
explain  the  location  of  so  many  liver  abscesses  at 
the  upper  portion  of  the  right  lobe  and  also  the 
occurrence  of  superficial  abscesses. 

It  is  very  doubtful  if  infection  often  occurs  by  way 
of  the  lymphatics,  although  amoebae  are  frequently 
noticed  in  this  location  in  sections  of  the  intestine. 
11 


162  PARASITIC  AMCEB^E  OF  MAN. 

Amoebic  abscesses  may  occur  in  other  regions  than 
the  liver;  the  amoeba?  reaching  the  various  organs  by 
way  of  the  blood-vessels.  Several  authors  have  re- 
ported the  finding  of  amoebae  in  the  blood,  and  un- 
doubtedly a  few  such  instances  are  authentic. 

The  Production  of  Dysentery  in  Susceptible 
Animals. — Since  the  first  description  of  amoeba? 
various  observers  have  succeeded  in  producing  dysen- 
tery in  susceptible  animals  by  rectal  injection,  or 
feeding  material  containing  amoebae  from  human 
sources  to  such  animals.  Unfortunately  all  the  work 
along  this  line  up  to  the  time  of  Schaudinn's  ob- 
servations is  inconclusive  as  regards  the  species  of 
amoebae  used  in  the  experiments,  for  while  there  is 
no  doubt  but  that  pathogenic  amoebae  were  employed 
we  are  uncertain  as  to  which  pathogenic  species  the 
experimenter  was  dealing  with,  and  for  this  reason 
these  experiments  cannot  be  used  as  proving  the 
pathogenicity  of  either  Entamceba  histolytica  or 
Entamceba  tetragena.  In  all  the  early  experiments 
in  which  positive  results  were  obtained  amoeba?  were 
used  which  answer  to  the  description  of  the  patho- 
genic species  now  recognized,  and  the  experiments 
proved  beyond  question  that  it  is  possible  to  produce 
typical  amoebic  dysentery  in  susceptible  animals  with 
such  amoeba?. 

Since  the  observation  of  Schaudinn  several  ob- 


AMOEBJ5  OF  THE  INTESTINAL  TRACT.     163 

servers  have  shown  that  Entamceba  Mstolytica  is 
capable  of  producing  the  lesions  of  dysentery  when 
injected  or  fed  to  susceptible  animals.  A  great 
amount  of  work  has  been  accomplished  by  Musgrave, 
and  Musgrave  and  Clegg,  in  experimental  dysentery, 
and  it  is  greatly  to  be  regretted  that  their  work 
cannot  be  used  in  proving  the  pathogenic  relation 
of  specific  amoebae  to  dysentery,  for  the  reason  that 
they  failed  to  recognize  species.  As  both  Entamceba 
histolytica  and  Entamceba  tetragena  occur  in  the 
Philippine  Islands,  these  authors  may  have  been  deal- 
ing with  both  species,  but  their  results  cannot  be 
definitely  attributed  to  either  one.  They  have,  how- 
ever, succeeded  in  producing  typical  dysentery  lesions 
in  the  intestine  of  monkeys,  and  abscess  of  the  liver 
in  the  same  animals. 

Schaudinn  experimented  upon  cats  and  concluded 
from  feeding  experiments  that  only  the  spores  of 
Ent amoeba  histolytica  were  capable  of  causing  dysen- 
tery in  these  animals.  He  thus  describes  his  ex- 
periments which  prove  this  point.  After  stating  that 
he  obtained  his  material  from  a  case  of  dysentery 
which  became  infected  in  China,  he  says: 

:<  From  this  case  I  took  a  small  quantity  of  feces, 
divided  it  into  three  parts,  dried  each  in  the  air,  and 
mixed  with  it  sufficient  water  for  about  20  crush 


164  PARASITIC  AMOEBAE  OF  MAN. 

preparations  under  cover  glasses.  These  preparations 
were  carefully  examined,  the  examination  being  con- 
ducted upon  a  mechanical  stage  and  requiring  many 
hours.  No  forms  resembling  the  cysts  of  Entamceba 
coli  were  found,  but  the  small  spores  of  Entamceba 
Mstolytica  were  noticed  in  large  numbers,  but  no 
vegetative  organisms  could  be  demonstrated.  The 
cover  glasses  were  then  removed,  the  feces  washed 
with  distilled  water,  and  ten  such  preparations  were 
mixed  with  enough  distilled  water  to  form  1  c.c.  of 
the  mixture.  The  feces  of  the  animal  to  be  ex- 
perimented upon,  a  healthy,  strong  young  cat,  was 
carefully  examined  for  amoebae  and  none  could  be 
demonstrated.  To  this  cat  I  gave  the  1  c.c.  prepara- 
tion mentioned  above,  mixing  it  with  milk.  On  the 
evening  of  the  third  day  the  cat  passed  bloody  mucoid 
feces  and  an  examination  showed  the  presence  of 
great  numbers  of  typical  Entamoeba  Mstolytica.  In 
the  afternoon  of  the  fourth  day  the  cat  perished. 
Dissection  showed  typical  ulcerous  dysentery  of  the 
large  intestine,  and  immigration  of  the  amoebse  into 
the  epithelium  could  be  easily  established. 

"  I  will  mention  yet  another  experiment  which 
goes  to  prove  that  the  permanent  spores  by  them- 
selves are  capable  of  producing  a  new  infection. 
The  feces  of  the  cats  developing  dysentery  con- 
tained only  vegetable  stages  of  the  amoebae,  no  spores 


AMOEBJ5  OF  THE  INTESTINAL  TRACT.     165 

being  found.  When  large  quantities  of  the  feces 
were  given  to  a  cat  it  remained  well  and  for  four 
weeks  showed  no  amoebae  in  its  feces.  It  was  then 
fed  with  the  remnant  of  the  dried  feces  used  in  the 
first  experiment,  which  contained  multitudes  of  the 
spores,  and  after  six  days  the  amoebae  began  to  appear 
in  the  feces.  Being  older  and  larger  than  the  other 
cat  it  proved  more  resistant  to  the  infection  and  did 
not  die  until  two  weeks  later.  The  autopsy  showed 
the  lesions  of  typical  amoebic  dysentery." 

These  experiments  of  Schaudinn  throw  a  flood 
of  light  upon  the  interpretation  of  the  negative  re- 
sults of  some  observers  who  have  worked  with  Enta- 
mceba  Mstolytica.  It  will  be  remembered  that  during 
the  active  stage  of  dysentery,  when  the  symptoms  are 
acute,  only  the  vegetative  stages  of  this  species  occur 
in  the  feces,  and  that  these  stages  do  not  cause  in- 
fection, while  the  spores  which  are  the  infective  agents 
in  this  species  only  occur  when  the  conditions  for 
vegetative  existence  are  unfavorable,  that  is,  when 
the  healing  process  has  begun.  If  these  points  be 
remembered  the  negative  results  obtained  by  feeding 
or  injection  of  feces  from  active  cases  of  dysentery 
can  be  explained,  as  in  such  cases  the  feces  do  not 
contain  the  spores.  If,  however,  the  feces  from  cases 
which  are  recovering  be  used  for  experimental  pur- 


166  PARASITIC  AMOEBAE  OF  MAN. 

poses  a  large  proportion  of  susceptible  animals  will 
develop  the  disease. 

The  uniformly  negative  results  reported  by  some 
authorities  are  explained  by  the  fact  that  they  were 
working  with  the  harmless  Entamceba  coli  or  with 
feces  containing  only  the  vegetative  stages  of  Enta- 
mceba  histolytica. 

Personal  Observations. — I  have  already  detailed 
the  negative  results  obtained  in  kittens  by  feeding 
experiments  and  rectal  injection  with  feces  contain- 
ing the  vegetative  and  encysted  stages  of  Entamceba 
coli.  These  experiments  have  been  repeated,  using 
the  feces  of  dysenteric  patients  containing  both  the 
vegetative  stages  and  the  spores  of  Entamoeba 
histolytica,  with  the  result  that  66  per  cent,  of  the 
kittens  experimented  upon  by  feeding  developed 
typical  amoebic  dysentery,  while  50  per  cent,  of  those 
in  which  rectal  injections  were  used  developed  the 
disease.  Control  tests  were  made  with  the  bacteria 
occurring  in  the  feces  and  with  feces  containing  Enta- 
moeba coli. 

Rectal  Injections. — This  is  the  method  which  has 
been  employed  by  most  investigators  of  this  subject, 
but  it  is  not  as  successful  as  the  feeding  experiments, 
only  50  per  cent,  of  the  animals  experimented  upon 
developing  the  disease.  Half-grown  kittens  were 
used,  about  5  c.c.  of  feces  containing  amoebae  being 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     167 

injected  into  the  rectum.  The  negative  results  may 
be  explained  by  the  absence  of  the  infective  spores, 
for  while  motile  amoebae  were  present  in  all  the  ma- 
terial used,  some  of  it  was  not  examined  for  spores, 
as  at  the  time  I  was  inclined  to  believe  that  the 
vegetative  stages  of  this  species  could  produce  in- 
fection. 

In  experimental  dysentery  produced  in  kittens  by 
the  rectal  injection  of  infected  material  the  lesions 
tend  to  be  localized  in  the  rectum,  and  are  not  so 
severe  as  when  the  infection  is  acquired  through  the 
mouth.  The  incubation  period  varies  from  6  days  to 
nearly  two  weeks,  being  longer  on  the  average  than 
in  the  feeding  experiments.  The  lesions  produced 
were  typical  of  those  occurring  in  amoebic  dysentery 
in  man  and  varied  in  extent  and  severity  with  the 
length  of  time  the  infection  lasted. 

It  is  not  necessary  at  this  time  to  give  in  detail  all 
of  the  autopsy  records  of  the  experimental  work  just 
mentioned,  but  I  shall  quote  here  in  full  the  autopsy 
record  of  a  case  of  dysentery  produced  in  a  kitten  by 
rectal  injection  as  it  is  characteristic  of  the  findings 
obtained  in  all  the  cases,  the  only  difference  being 
in  the  severity  and  extent  of  the  lesions.  This  kitten 
was  given  a  rectal  injection  of  5  c.c.  of  feces  from  a 
case  of  dysentery  contracted  in  the  Philippine  Islands, 
the  date  of  injection  being  October  19.  Upon 


168  PARASITIC  AMGEBJS  OF  MAN. 

October  30,  the  animal  showed  amosbse  in  the  feces, 
soon  developed  a  bloody  diarrhoea  and  was  killed  upon 
November  21,  the  infection  having  lasted  approxi- 
mately 30  days. 

Kitten  1. — Body  that  of  a  half -grown  kitten,  very 
greatly  emaciated.  The  abdomen  is  greatly  distended 
with  gas.  The  mucous  membrane  of  the  anus 
appears  swollen  and  a  considerable  amount  of  blood- 
stained mucus  is  adherent  to  it.  The  subcutaneous 
fat  has  almost  entirely  disappeared  and  the  muscles 
appear  dry  and  atrophied.  The  pleural  cavities 
are  free  from  fluid  and  the  lungs  appear  normal. 
The  heart  is  greatly  congested  and  contains  red  clots 
in  all  the  chambers.  The  liver  is  hypertrophied, 
deeply  congested,  and  marked  albuminoid  degenera- 
tion is  present,  but  there  is  no  trace  of  abscess  forma- 
tion. The  kidneys  are  congested  and  upon  section 
present  the  usual  lesions  of  an  acute  parenchymatous 
nephritis.  The  omentum  contains  a  small  amount 
of  fat  and  is  not  inflamed.  The  bladder  is  filled 
with  urine. 

The  intestines  are  greatly  dilated  with  gas  and 
fluid.  Upon  external  examination  the  large  intes- 
tine appears  swollen,  is  grayish  in  color,  with  small, 
darker  colored  areas  scattered  along  it.  Upon  open- 
ing the  large  intestine  the  mucous  membrane  of  the 
rectum  is  found  considerably  swollen  and  inflamed, 


AM(EBJE  OF  THE  INTESTINAL  TRACT.     169 

but  no  ulcerations  are  present.  Above  the  rectum 
for  a  distance  of  about  10  cm.  the  mucous  membrane 
is  very  much  swollen  and  cedematous,  bright  red  in 
color,  and  between  the  folds  a  considerable  amount 
of  pus  can  be  seen.  For  a  distance  of  about  4  cm. 
from  the  upper  end  of  the  large  intestine,  the  mucous 
membrane  is  inflamed,  being  red,  swollen,  and 
oedematous.  In  this  area  there  are  numerous  ulcera- 
tions, covered  in  with  bloody  mucus ;  they  are  of  small 
size,  somewhat  irregular  in  shape,  and  extend,  in  most 
instances,  to  the  submucosa,  although  there  are  a 
few  which  extend  to  the  muscular  coat  of  the  in- 
testine; the  edges  are  undermined  and  many  of  the 
ulcers  are  covered  with  necrotic  tissue,  brownish  yel- 
low in  color,  which  has  to  be  removed  in  order  to 
expose  them.  A  few  of  the  ulcers  communicate  be- 
neath the  mucous  membrane.  The  small  intestine 
shows  a  rather  severe  acute  enteritis  and  the  stomach 
an  acute  gastritis. 

In  this  case  the  greatest  number  of  ulcerations 
occurred  near  the  ileocaecal  valve,  while  the  rectum 
escaped ;  this  is  most  unusual,  as  in  the  other  success- 
ful experiments  by  rectal  injection  the  ulcerations 
were  generally  confined  to  the  rectum,  at  most  in- 
vading the  intestine  for  a  short  distance  only  above 
the  rectum. 

At  autopsy  actively  motile  Entamceba  histolytica 


170  PARASITIC  AMOEBAE  OF  MAN. 

could  be  easily  demonstrated  in  smears  made  from 
the  intestine,  being  most  numerous  where  the  lesions 
were  most  severe,  although  every  part  of  the  large 
intestine  showed  infection  with  this  parasite.  Smears 
from  the  small  intestine  were  negative  for  amoebae. 

The  clinical  symptoms  in  this  case  were  similar 
to  those  occurring  in  all  the  kittens  developing  amoebic 
dysentery  and  to  those  occurring  in  man.  The  first 
symptom  observed  was  diarrhoea,  the  stools  being 
frequent  and  free  from  blood  at  first,  but  soon  be- 
coming mucoid  and  bloody  and  containing  numerous 
amoebae.  Fever  was  generally  present  and  emaciation 
was  rapid.  In  this  kitten  the  diarrhoea,  after  per- 
sisting for  several  days,  ceased,  and  a  period  of  con- 
stipation intervened,  covering  two  or  three  days,  after 
which  the  dysenteric  symptoms  returned  and  from 
that  time  until  the  animal  was  killed  the  bowel  move- 
ments varied  in  number  from  6  to  10  a  day  and 
emaciation  became  extreme.  Amoeba?  could  always 
be  demonstrated  in  the  feces  after  the  initial  diarrhoea. 

The  lesions  produced  in  kittens  by  the  rectal  in- 
jection of  material  containing  Entamceba  histolytica 
are  perfectly  typical  of  the  lesions  of  amoebic  dysen- 
tery in  man,  making  allowance,  of  course,  for  the 
lesser  extent  of  the  surface  involved. 

Feeding  Experiments. — The  most  successful 
results  in  producing  dysentery  in  kittens  with  this 


AMCEB^E  OF  THE  INTESTINAL  TRACT.     171 

species  of  amoeba  are  obtained  by  feeding  the  animals 
with  infected  fecal  material.  With  this  method  I 
have  been  successful  in  producing  the  disease  in  8  out 
of  12  kittens  or  66+  per  cent,  and  in  every  case  have 
demonstrated  Entamceba  Mstolytica  in  the  feces  and 
in  sections  of  the  diseased  intestines.  The  method  of 
experimentation  was  as  follows: 

The  kittens  were  starved  for  24  hours,  at  the  end 
of  which  time  they  were  given  milk  containing  about 
5  c.c.  of  feces  in  which  both  motile  amoeba?  and  spores 
were  present;  the  animals  did  not  object  to  taking 
the  mixture  if  they  were  kept  without  food  for  this 
length  of  time,  but  unless  this  was  done  they  almost 
invariably  refused  the  infected  milk.  After  feeding, 
the  animals  were  placed  in  cages  and  carefully 
observed. 

When  successful  the  symptoms  consisted  of  diar- 
rhoea, rapid  emaciation,  loss  of  appetite  and  strength, 
severe  tenesmus,  the  animals  appearing  much  dis- 
tressed while  voiding  the  feces,  and  finally  death  from 
exhaustion.  The  feces  were  blood-stained,  containing 
much  mucus  and  multitudes  of  motile  amoebae. 

The  period  of  incubation  varied  from  7  to  11 
days,  the  average  being  8  days,  so  that  it  may  be 
stated  that  the  period  of  incubation  is  shorter  in 
feeding  experiments  than  when  the  infected  material 
is  introduced  per  rectum.  In  two  of  the  animals 


172  PARASITIC  AMOEBA  OF  MAN. 

short  periods  of  constipation  occurred  after  the 
initial  diarrhoea,  lasting  a  day  or  two,  but  they  were 
always  succeeded  by  diarrhoea  with  the  passage  of 
typical  dysenteric  stools. 

In  order  to  illustrate  the  lesions  produced  in  these 
animals  the  following  autopsy  records  of  two  of 
them  are  inserted,  as  these  are  typical  of  the  lesions 
observed  in  the  other  kittens. 

Kitten  3. — This  kitten  was  fed  once  with  feces 
containing  Entamceba  Tilstolytica  and  seven  days 
later  developed  diarrhoea,  the  feces  containing  blood 
and  mucus,  as  well  as  numerous  motile  amoebse.  At 
the  end  of  two  weeks  it  died,  having  presented  severe 
symptoms  of  amoebic  dysentery  during  this  time. 

Autopsy. — Body  that  of  a  half -grown  kitten,  very 
greatly  emaciated.  Subcutaneous  fat  entirely  absent, 
and  muscles  dry  and  much  atrophied.  The  abdominal 
cavity  is  free  from  fluid  and  the  intestines  appear 
normal  externally.  The  pleural  cavities  are  free  from 
fluid  and  the  heart  and  lungs  appear  normal.  The 
liver  is  brownish-red  in  color  externally,  with  irregular 
yellow  mottlings.  There  is  a  small  abscess  present  at 
the  dome  of  the  right  lobe,  measuring  0.25  cm.  in 
diameter,  showing  very  distinctly  through  the  capsule 
of  the  organ.  Upon  section  of  the  liver  the  cut  sur- 
face appears  greatly  congested,  the  lobules  are  dis- 
tinct, and  no  abscesses  are  found  other  than  the  one 


AM(EB,£  OF  THE  INTESTINAL  TRACT.     173 

mentioned.     The  gall  bladder  appears  normal.    The 
kidneys  appear  enlarged  and  congested  and  upon 
section  show  an  acute  congestion,  with  some  thicken- 
ing of  the  cortex.     Externally  the  large  intestine 
appeared  slightly,  if  at  all,  congested,  although  the 
walls  were  markedly  thickened.     Upon  opening  the 
large  intestine  it  was  found  filled  with  fecal  material 
mixed  with  a  large  amount  of  pus,  and  blood-stained 
mucus.    About  1  cm.  from  the  anus,  which  was  blood- 
stained and  covered  with  mucus,  there  was  an  area 
measuring  4  cm.  in  length,  presenting  the  typical 
lesions  of  amoebic  dysentery,  as  they  are  observed  in 
man.     The  entire  mucous  membrane  was  swollen, 
congested,  and  oedematous.    Numerous  nodular  areas 
projected  into  the  lumen  of  the  intestine,  which, 
when  incised,  were  found  filled  with  a  glairy  ma- 
terial containing  hundreds  of  Entamceba  histolytica. 
There  were  also  numerous  ulcerations,  more  or  less 
irregular  in  shape,  with  thickened  and  undermined 
edges;    many  were  covered  in  with  necrotic  tissue, 
which,  upon  being  removed,  showed  that  the  floor  of 
the  ulcer  was  formed  by  the  muscular  coat  of  the 
intestine.    Many  of  these  ulcers  communicated  with 
one  another  beneath  the  mucous  membrane,  and  most 
of  them  had  penetrated  to  the  muscular  coat.     The 
remainder  of  the  large  intestine  presented  numerous 
ulcerations,  typical  of  those  seen  in  the  intestine  of 


174  PARASITIC  AMOEBAE  OF  MAN. 

patients  who  have  died  of  amoebic  dysentery.  The 
lesions  were  most  marked  just  below  the  ileocascal 
valve,  where  large  areas  of  the  mucous  membrane 
had  been  destroyed,  the  muscular  coat  of  the  intestine 
being  exposed. 

Kitten  5. — This  kitten  was  fed  with  milk  contain- 
ing Entamceba  Ustolytica  several  times  before  dysen- 
tery developed.  The  period  of  incubation  was  eight 
days  from  the  date  of  the  last  feeding,  but  from  that 
time,  until  it  was  killed,  three  weeks  afterward,  the 
animal  presented  the  symptoms  of  amoebic  dysentery, 
there  being  gradual  loss  of  appetite,  emaciation,  and 
a  diarrhoeal  discharge,  containing  blood  and  mucus, 
with  numerous  motile  Entamoeba  histolytica. 

Autopsy. — Body  that  of  a  half-grown  kitten, 
much  emaciated.  Subcutaneous  fat  entirely  absent, 
and  muscles  much  atrophied.  The  pleural  cavities 
were  free  from  fluid  and  the  lungs  and  heart  appeared 
normal  save  for  congestion.  Upon  opening  the  ab- 
dominal cavity  the  small  intestine  appeared  congested 
externally.  The  liver  is  hypertrophied  and  greatly 
congested.  The  kidneys  are  congested  and  enlarged 
and  upon  section  showed  the  lesions  of  an  acute 
parenchymatous  nephritis.  The  large  intestine  was 
dark  gray  in  color  externally,  and  was  considerably 
thickened,  especially  toward  the  rectum.  Upon  open- 
ing the  intestine  it  was  found  to  contain  much  fecal 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     175 

material,  mixed  with  blood,  mucus,  and  pus.  Com- 
mencing at  the  rectum  and  extending  for  about  half 
the  length  of  the  large  intestine,  the  mucous  mem- 
brane was  greatly  swollen,  bright  red  in  color,  and 
contained  numerous  ulcers.  The  majority  of  the 
ulcers  were  spherical  in  shape,  the  edges  were  under- 
mined and  greatly  thickened,  and  many  were  covered 
in  with  necrotic  tissue.  Upon  removing  this  necrotic 
material  the  base  of  the  ulcer  is  found  to  be  formed 
by  the  muscular  coat  of  the  intestine.  The  ulcers 
present  were  typical  of  the  amoebic  ulcerations  seen 
in  the  intestine  of  man  in  every  respect.  The  re- 
mainder of  the  large  intestine  was  black  in  color  and 
gangrenous,  the  mucous  membrane  having  been 
almost  entirely  destroyed,  exposing  the  muscular  coat 
throughout  this  portion  of  the  intestine.  About  4  cm. 
below  the  ileocsecal  valve  there  was  a  small  perfora- 
tion measuring  about  one-sixth  cm.  in  diameter. 

I  believe  that  it  must  be  evident  to  anyone  from 
the  autopsy  records  given  that  the  lesions  produced 
in  kittens  by  feeding  them  with  material  containing 
Entamceba  histolytica  are  typical  of  the  lesions  of 
amoebic  dysentery  in  man.  The  examination  of  sec- 
tions of  the  diseased  intestines  showed  the  same  micro- 
scopic pathology  observed  in  sections  from  the 
dysenteric  intestine  of  man,  and  the  amoebae  were 
demonstrated  in  the  same  situations  within  the  in- 


176  PARASITIC  AMCEBJE  OF  MAN. 

testinal  coats.  The  amoebae  observed  in  the  feces  and 
in  the  sections  of  intestine  presented  the  morpho- 
logical characteristics  of  Entamceba  Mstolytica  in 
every  case. 

Control  Experiments. — All  the  kittens  experi- 
mented upon  were  very  carefully  examined  for 
several  days  prior  to  infecting  them  for  the  presence 
of  amoebae,  and  all  were  found  negative,  so  that  there 
can  be  no  question  of  a  previous  amoebic  infection. 

That  amoebae  produced  the  lesions  of  dysentery 
observed  in  the  kittens,  and  not  the  bacteria  occur- 
ring with  the  amoebae  in  the  feces,  was  proven  by 
using  pure  cultures  of  the  various  bacteria  from  the 
feces  for  feeding  and  injections,  and  mixed  cultures 
of  all  the  bacteria  that  could  be  cultivated  were  also 
used  in  the  same  manner,  but  in  no  case  did  diarrhoea 
or  dysentery  result.  While  there  were  probably  bac- 
teria present  which  could  not  be  cultivated,  I  do  not 
believe  that  this  militates  against  the  conclusion  that 
Entamceba  histolytica  was  the  cause  of  the  lesions 
produced. 

That  the  bacteria  occurring  in  the  feces  of  amoebic 
dysentery  cases  are  not  the  cause  of  the  lesions  ob- 
served in  experimental  animals  was  previously  proven 
conclusively  by  Harris.  He  injected  4  dogs  per 
rectum  with  fecal  material  containing  amoebae  and 
in  all  of  them  the  injection  was  followed  by  dysentery. 
In  2  cases  abscess  of  the  liver  was  found  after  death, 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     177 

the  contents  of  the  abscesses  containing  amoebae.  In 
order  to  control  his  experiments  he  believed  it  was 
necessary  to  cultivate  the  bacteria  found  in  the  feces 
of  dysenteric  patients  and  to  inject  these  separately 
and  together.  Regarding  these  experiments  he  says : 

"  Cultures  were  made  from  the  feces  of  the  same 
individuals  whose  discharges  had  been  used  to  suc- 
cessively produce  dysentery,  and  these  were  then  in- 
jected into  the  intestines  of  four  puppies.  There  was 
absolutely  no  effect  produced.  It,  therefore,  seems 
unreasonable  to  conclude  that  the  germ  that  produces 
the  disease  is  a  bacterium;  or,  at  any  rate,  it  seems 
fairly  certain  that  it  cannot  be  an  organism  that 
develops,  or  even  lives,  in  the  culture  media  ordinarily 
employed.  As  neither  of  these  suppositions  appears 
at  all  probable,  and  as  the  amoeba  was  the  only  other 
living  organism  found  in  the  feces,  that  was  probably 
absent  from  the  cultures,  it  seems  logical  to  suppose 
that  this  parasite  is  the  cause  of  any  morbid  state 
that  the  injection  of  these  discharges  may  give  rise 
to.  This  view  is  supported  by  the  fact  that  the  amoebae 
are  abundantly  present  in  and  around  the  ulcers  that 
are  found  in  the  intestines  of  the  dogs  suffering  from 
experimental  dysentery,  and  it  does  not  appear  un- 
reasonable to  say  that  the  proof  is  now  fairly  clear 
that  these  organisms  are  in  reality  the  causative 
agents  in  chronic  dysentery." 


12 


178  PARASITIC  AMCEB^E  OF  MAN. 

In  one  of  the  infected  kittens  amoebic  abscess  of 
the  liver  occurred  and  the  pus  from  this  abscess  was 
sterile  except  for  the  presence  of  a  few  amoebse,  while 
these  parasites  were  also  demonstrated  in  sections  of 
the  abscess  wall.  This  observation  is  almost  conclusive 
of  the  etiological  relationship  of  Entamoeba  Msto- 
lytica  to  abscess  of  the  liver  complicating  dysentery. 

Control  experiments  were  also  made  with  Enta- 
moeba eoli  with  a  negative  result  in  every  case. 

My  experiments  regarding  the  production  of 
dysentery  in  kittens  by  Entamoeba  histolytica  have 
been  confirmed  by  Werner,  working  at  the  Sailors' 
Hospital  in  Hamburg.  He  experimented  with  two 
strains  of  Entamoeba  histolytica,  only  one  of  which 
he  found  infective.  He  was  able  to  produce  dysentery 
in  cats  with  this  strain,  but  found  that  after  six 
passages  the  organism  lost  its  virulence.  The  incuba- 
tion period  varied  from  4  to  13  days,  the  average 
being  9  days.  Of  six  cats  infected  with  this  species 
four  died,  the  duration  of  the  disease  varying  from 
7  to  24  days,  the  average  being  15  days.  The 
animals  were  infected  per  rectum.  Werner  states 
that  the  lesions  were  typical  of  amoebic  dysentery  and 
were  always  confined  to  the  colon,  especially  the 
lower  portion.  Guinea-pigs  and  rats  were  found  to 
be  resistant  to  infection  with  this  parasite. 

From  the  evidence  which  has  been  submitted  it 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     179 

appears  to  me  to  be  impossible  to  conclude  otherwise 
than  that  Entamceba  Mstolytica  is  the  cause  of  a  form 
of  amoebic  dysentery.  The  character  of  the  lesions 
present  in  this  condition,  the  constant  association  of 
this  species  with  the  lesions,  and  the  production  of 
similar  lesions  in  susceptible  animals,  with  infected 
material,  I  consider  conclusive  proof  that  this  para- 
site causes  amoebic  dysentery  in  man. 

Cultivation. — The  subject  of  the  cultivation  of 
this  species  of  amoeba  will  be  considered  in  the  section 
dealing  with  Entamceba  tetragena,  but  I  will  here 
state  that  I  have  tried  all  the  methods  recommended 
by  those  who  claim  to  have  been  successful  in  cul- 
tivating these  organisms,  but  have  never  been  able 
to  secure  growth  upon  artificial  media.  In  a  recent 
article  Noc  appears  to  have  been  successful  in  cul- 
tivating an  amoeba  occurring  in  dysentery  in  Cochin 
China,  which  bears  a  very  close  resemblance,  as  in- 
dicated in  his  description,  to  Entamoeba  histolytica, 
or  tetragena,  but  up  to  the  present  time  his  observa- 
tions have  not  been  confirmed. 

ENTAM(EBA  TETRAGENA.    Viereck,  1907. 

In  1907,  Viereck  described  an  amoeba  occurring 
in  patients  suffering  from  dysentery  contracted  in 
Africa  which  he  considered  a  new  species,  and  to 
which  he  gave  the  name  Entamoeba  tetragena.  Dur- 
ing the  same  year  an  independent  description  of  the 


180  PARASITIC  AMCEB^E  OF  MAN. 

same  organism  was  published  by  Hartmann  and 
Prowazek,  who  named  it  Entamceba  africana.  As 
the  description  of  Viereck  was  published  before  that 
of  Hartmann  and  Prowazek  the  name  applied  to  the 
organism  by  Viereck  must  remain  as  the  proper 
zoological  name  of  this  species.  The  observations  of 
the  authors  mentioned  have  been  confirmed  by  Bensen 
and  others  and  it  is  now  generally  accepted  that  this 
species  is  the  cause  of  a  form  of  amoebic  dysentery. 
At  the  time  that  I  had  the  opportunity  of  study- 
ing amoeba?  in  soldiers  returning  from  the  Philippines 
to  San  Francisco,  and  in  the  Philippines,  the  species 
now  known  as  Entamceba  tetragena  had  not  been 
described.  I  had  several  times  observed  amoeba?  which 
could  not  be  considered  typical  of  either  Entamceba 
histolytica  or  Entamceba  coli  in  that  they  possessed 
a  distinct  ectoplasm,  a  well  defined  nucleus  contain- 
ing much  chromatin,  and  reproduced  by  simple 
division  and  the  formation  of  cysts  containing  four 
daughter  amoebae.  I  observed  these  organisms  both 
in  soldiers  suffering  from  dysentery  contracted  in 
the  Philippines  and  in  the  natives  of  those  islands, 
and  until  the  description  by  Viereck  of  Entamceba 
tetragena  I  considered  them  as  atypical  forms  of  the 
other  species.  Upon  looking  over  my  notes  of  cases 
observed  during  the  past  seven  years  I  find  frequent 
notations  of  the  occurrence  of  an  amoeba  correspond- 


OF  THE  INTESTINAL  TRACT.     181 

ing  with  Entamceba  tetragena  in  dysenteric  patients 
returning  from  the  Philippine  Islands,  and  during 
the  last  winter  while  demonstrating  these  parasites 
to  the  class  at  the  Army  Medical  School,  my  atten- 
tion was  called  to  amoebae  in  one  of  the  specimens 
which  answered  to  the  description  of  this  species. 
The  amoebae  in  this  instance  were  obtained  from  a 
discharged  soldier  who  had  contracted  dysentery  in 
the  Philippine  Islands  and  who  had  suffered  from 
many  recurrences.  This  observation  taken  in  con- 
junction with  my  previous  records  upon  a  similar 
amoeba  observed  in  soldiers  returning  from  the  Philip- 
pines and  in  natives  of  the  Philippine  Islands  con- 
vinces me  that  Entamceba  tetragena  is  a  not  infre- 
quent cause  of  dysentery  in  those  Islands.  I  have 
also  found  this  species  in  material  sent  me  by  Captain 
Siler  of  the  Medical  Corps,  U.  S.  Army,  from  cases 
of  dysentery  originating  in  Illinois. 

Much  of  the  confusion  and  difficulty  regarding  the 
differentiation  of  Entamceba  coli  and  Entamceba  his- 
tolytica  has  undoubtedly  been  due  to  the  presence  in 
many  cases  of  this  third  species  which  possesses  mor- 
phological features  common  to  both  of  the  other 
species.  Thus,  while  Entamceba  Mstolytica  possesses: 
an  ill  defined  nucleus  and  does  not  form  cysts  as 
does  Entamceba  coli,,  yet  in  certain  cases  of  dysentery 
amoebae  may  be  observed  in  which  there  is  a  well  de- 


182  PARASITIC  AMOEBJS  OF  MAN. 

fined  nucleus,  but  cysts  are  formed  only  differing 
from  those  of  Entamceba  coli  in  the  presence  of  four 
instead  of  eight  daughter  amoebae.  The  identification 
of  this  latter  form  as  Entamceba  tetragena  explains 
the  apparent  contradiction  and  will,  I  am  sure,  render 
the  classification  of  the  amoebae  found  in  man  much 
less  difficult. 

GEOGRAPHICAL  DISTRIBUTION. — The  geographical 
distribution  of  Entamceba  tetragena  has  not  been 
thoroughly  studied.  The  observations  of  Viereck 
and  of  Hartmann  and  Prowazek  prove  that  it  occurs 
in  East  Africa,  Farther  India,  China  and  probably 
in  other  countries  of  the  Far  East.  My  own  ob- 
servations show  that  it  occurs  in  the  Philippine 
Islands,  and  it  is  very  probable  that  it  is  a  frequent 
cause  of  dysentery  in  many  of  the  islands  of  the 
Pacific.  This  species  is  also  found  in  South  America, 
and  it  is  not  unlikely  that  it  may  be  a  frequent  cause 
of  dysentery  in  certain  portions  of  the  United  States. 

MORPHOLOGY. — Entamceba  tetragena  resembles 
in  certain  features  of  its  morphology  both  coli  and 
Mstolytica.  It  consists  of  a  mass  of  protoplasm  con- 
taining a  well  defined  nucleus,  is  actively  motile,  and 
reproduces  by  simple  division  and  by  cysts  containing 
four  daughter  amoebae.  I  had  an  opportunity  of 
studying  this  organism  in  f eces  from  the  case  already 
mentioned,  and  can  confirm  the  description  given  of 
it  by  Viereck  and  Hartmann  and  Prowazek. 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     183 

Size. — This  parasite  is  slightly  smaller  than  Enta- 
mceba  histolytica  measuring  from  15  to  45  microns 
in  diameter,  while  the  cysts  measure  From  7  to  12 
microns  in  diameter.  The  size  is  of  no  value  in  the 
differentiation  of  the  species  because  the  organisms 
resemble  in  this  respect  both  Entamoeba  coli  and 
Entamoeba  histolytica. 

Shape. — When  at  rest  the  organism  is  spherical 
in  shape,  but  great  variations  in  its  contour  are  ob- 
served when  it  is  in  motion. 

The  Protoplasm. — As  in  other  amoeba?  the  ap- 
pearance of  the  protoplasm  varies  with  the  age  of 
the  organism.  The  very  young  amoebae  show  a 
distinctly  granular  protoplasm  in  which  there  is  a 
well  defined  nucleus.  In  the  older  amoebae  the  proto- 
plasm appears  still  more  granular  and  in  almost 
every  instance  a  definite  nucleus  having  a  strong 
nuclear  membrane  is  observed.  The  ectoplasm  can- 
not be  distinguished  when  the  parasite  is  motionless, 
but  it  is  very  distinct  when  motility  is  present. 

The  Cytoplasm. — When  fully  developed  the  cyto- 
plasm of  Entamoeba  tetragena  presents  two  well  de- 
fined portions,  an  outer,  the  ectoplasm,  and  an  inner, 
the  endoplasm.  This  distinction  is  not  evident  unless 
the  organism  is  moving,  but  in  such  instances  it  will 
be  observed  that  the  pseudopodia  formed  of  ecto- 
plasm are  composed  of  material  distinctly  different 


184  PARASITIC  AMCEBJS  OF  MAN. 

from  that  composing  the  endoplasm.  The  ectoplasm 
is  hyaline  in  appearance  and  resembles  that  of  Enta- 
mceba  histolytica,  being  very  refractile  and  glass- 
like.  I  have  not  been  able  to  distinguish  any  differ- 
ence in  the  appearance  of  the  ectoplasm  of  this  species 
and  that  of  histolytica.  Under  high  power  the 
ectoplasm  appears  to  be  composed  of  multitudes  of 
minute  granules  suspended  in  a  homogeneous  plastic 
substance. 

The  endoplasm  of  this  species  has  a  grayish  ap- 
pearance and  in  the  fully  developed  organism  ap- 
pears to  be  composed  of  various  sized  granules  and 
contains  within  it  bacteria,  crystals,  and  in  most  in- 
stances, one  or  more  vacuoles.  In  this  species  the 
number  of  vacuoles  is  not  as  great  as  in  Entamceba 
histolytica,  and  not  infrequently  organisms  are 
observed  in  which  no  vacuoles  are  present.  The 
vacuoles  are  not  contractile  so  far  as  I  have  been 
able  to  observe. 

The  Nucleus. — The  nucleus  of  this  species  is  one 
of  its  most  characteristic  features.  It  is  compara- 
tively large  and  is  always  well  defined.  The  struct- 
ure may  be  described  as  follows:  Externally  there 
is  a  well  marked  nuclear  membrane,  sharply  dis- 
tinguished from  the  endoplasm,  and  very  refractile; 
internally  there  is  a  large  amount  of  chromatin  situ- 
ated upon  the  inner  side  of  the  nuclear  membrane 


B 


FIG.  XXII. — A,  Entamoeba  tetragena.  Living  vegetative  form  showing 
character  of  the  nucleus  and  the  well-defined  ectoplasm.  (After  Hartmann.) 
B,  Entamoeba  tetragena,  stained,  showing  nuclear  membrane,  karyosome, 
and  centriole.  X  1400.  (After  Hartmann.)  C  and  D,  two  nuclei  of  Ent- 
amoeba tetragena  greatly  enlarged,  showing  the  centriole  and  karyosome 
and  the  reticulative  appearance  due  to  cyclical  changes  in  the  latter. 
(After  Hartmann.) 


^  \ 

,**' 


,V-i* 


FIG.  XXIII. —  Various  stages  in  the  development  of  Entamoeba  tctra- 
gena.  (After  Hartmann.)  A,  a  parasite  showing  chromidia  and  beginning 
division  of  the  nucleus,  the  centriole  having  disappeared;  B,  organism  show- 
ing division  of  the  nucleus;  C,  a  cystic  form  in  the  uninuclear  stage;  D, 
encysted  form  containing  two  nuclei;  E,  encysted  form  containing  four 
nuclei. 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     185 

in  nodular  masses  and  distributed  throughout  the 
nuclear  substance  in  the  form  of  a  network  of  minute 
oval  or  irregular  granules ;  the  karyosome  is  large  and 
has  a  centriola  or  centrosome,  surrounded  by  a  clear 
area,  and  in  some  instances  two  or  three  con- 
centric clear  areas  surround  the  centriola;  the 
latter  is  spherical  in  shape  and  varies  in  size  from 
a  minute  dot  to  a  comparatively  large  spherical  mass. 

During  the  reproductive  cycle  certain  changes 
occur  in  the  karyosome  by  reason  of  the  rearrange- 
ment of  the  chromatin  which  lead  to  marked  differ- 
ences in  the  appearance  of  the  nucleus  at  different 
stages  of  development. 

Vacuoles  and  Contained  Bodies. — Vacuoles  are 
not  as  constantly  present  in  this  species  as  in  Enta- 
mceba  histolytica.  Very  frequently  well-grown 
organisms  are  observed  in  which  a  vacuole  is  absent, 
while  they  are  never  as  numerous  as  they  may  be  in 
the  latter  species. 

This  organism  is  phagocytic  for  red  blood  cor- 
puscles and  in  feces  containing  blood  the  amoebae 
are  usually  observed  to  have  engulfed  the  red  cells. 
So  far  as  I  have  been  able  to  observe  they  are  as 
actively  phagocytic  as  Entamceba  histolytica. 

The  cytoplasm  of  this  species  does  not  contain 
the  small  oval  bodies  which  are  so  frequently  found 
in  histolytica,  although  during  certain  stages  of  re- 


186  PARASITIC  AMOEBAE  OF  MAN. 

production  the  collections  of  chromatin  which  form 
the  nuclei  of  the  young  amoebae  may  appear  as  round 
or  oval,  highly  refractile  masses,  within  the  endo- 
plasm. 

As  in  other  parasitic  amoebae  the  endoplasm  con- 
tains bacteria  of  various  kinds,  crystals  and 
extraneous  matter  derived  from  the  feces. 

Motility. — What  has  been  said  regarding  the 
motility  of  Entamceba  histolytica  applies  equally  as 
well  to  this  species.  The  same  forms  of  motility  are 
observed  and  I  have  not  been  able  to  detect  any 
difference  in  the  rate  of  motility,  both  of  these  species 
being  very  actively  motile  as  compared  with  Enta- 
moeba  coll. 

The  pseudopodia  which  are  projected  when  the 
organisms  are  in  motion  are  composed  entirely  of 
ectoplasm  and  are  generally  more  or  less  finger-like 
in  shape,  having  blunt  extremities.  In  this  species 
the  pseudopodia  are  very  clearly  distinguished  from 
the  rest  of  the  organism  and  are  clear  and  glass-like 
in  appearance.  When  the  organisms  are  actively 
motile  the  endoplasm  very  quickly  flows  into  the 
pseudopodia  so  that  in  some  instances  it  is  rather 
difficult  to  distinguish  the  boundary  line  between 
these  two  portions  of  the  cytoplasm. 

Stained  Preparations. — This  parasite  may  be 
stained  by  any  of  the  methods  which  have  been  recom- 


OF  THE  INTESTINAL  TRACT.     187 

mended  for  staining  amoebae.  The  Wright  stain  and 
the  iron  haematoxylin  methods  give  the  best  results, 
and  if  the  latter  is  used  the  specimens  should  be  fixed 
while  wet. 

Entamoeba  tetragena  does  not  show  the  difference 
in  the  staining  reactions  of  the  ecto-  and  endoplasm 
with  Wright's  stain  which  is  so  characteristic  of  well 
stained  preparations  of  Entamoeba  histolytica.  In 
this  species  the  cytoplasm  stains  a  well  marked  blue, 
when  Wright's  stain  is  used,  while  the  nucleus,  being 
rich  in  chromatin,  stains  a  ruby  red  or  dark  violet 
color.  In  organisms  which  are  undergoing  division 
the  nucleus  is  often  observed  to  be  divided  into  two 
spherical  reddish  bodies  connected  by  delicate  pinkish 
strands  of  chromatin  while  further  division  is  some- 
times indicated  by  the  presence  of  three  or  four  masses 
of  red  stained  chromatin  situated  within  the  endo- 
plasm. This  species  does  not  show  the  division  of 
the  chromatin  into  delicate  fibriles,  distributed 
throughout  the  cytoplasm,  as  does  Entamoeba  his- 
tolytica,  but  elongated  spindle-shaped  masses  of  this 
substance  are  frequently  observed  in  amoebae  under- 
going reproduction. 

Reproduction. — This  species  of  amoeba  reproduces 
by  simple  division  and  by  cyst  formation  with  the 
production  within  the  cyst  of  four  young  amoebae. 
Simple  division  is  preceded  by  the  mitotic  division 


188  PARASITIC  AMCEBJE  OF  MAN. 

of  the  nucleus,  followed  by  the  division  of  the  cyto- 
plasm, resulting  eventually  in  the  production  of  two 
amoebse.  Mitosis  is  of  a  primitive  type,  but  in  well 
stained  specimens,  especially  if  the  iron  hsematoxylin 
method  be  used,  mitotic  figures  are  frequently 
observed.  The  karyosome  first  divides,  followed  by 
the  division  of  the  nucleus. 

Reproduction  in  a  cyst  is  preceded  by  cyclical 
changes  in  the  nucleus.  These  changes  have  been 
well  described  by  Hartmann,  who  states  that  in  no 
other  species  of  amoeba  has  he  observed  such  clear 
cyclical  changes  in  the  karyosome.  Wet-fixed  prepa- 
rations, in  sublimate  alcohol,  and  stained  with  iron 
hsematoxylin  should  be  used  in  studying  the  nuclear 
changes. 

Prior  to  encystment  the  karyosome  apparently 
becomes  differentiated  into  distinct  portions,  a  net- 
work composed  of  refractile  fibres  which  contains 
within  it  a  clear  fluid-like  substance.  The  clear  area 
surrounding  the  centriole  gradually  disappears  and 
prior  to  division  the  nucleus  appears  to  be  composed 
of  a  delicate  network  upon  which  are  arranged  minute 
masses  of  chromatin  which  appear  highly  refractile 
in  the  living  specimen. 

As  these  changes  are  occurring  in  the  nucleus  the 
organism  extrudes  all  foreign  matter,  becoming  hya- 
line in  appearance  and  spherical  in  shape.  The  nucleus 


FIG.  XXIV. —  Various  stages  in  the  life-cycle  of  Entamoeba  tetragena. 
(After  Viereck.)  1,  vegetative  form ;  2  and  3,  forms  showing  division  of  the 
nucleus;  4  to  12,  stages  in  nuclear  reduction  and  development.  Note  the 
large  amount  of  chromatm  during  some  of  the  stages.  Figs.  11  and  12  are 
encysted  forms. 


AMQEB.E  OF  THE  INTESTINAL  TRACT.     189 

then  divides  into  two  well  defined  portions,  each  con- 
taining a  large  mass  of  chromatin  and  several  small 
granules  of  the  same  substance,  the  large  mass  being 
situated  at  one  side  of  the  nucleus.  The  chromatin 
apparently  increases  in  amount  during  this  process 
of  division  and  some  of  it  is  distributed  to  the  cyto- 
plasm. A  cyst  wall  is  gradually  developed,  and  the 
two  nuclei  each  divide,  thus  forming  the  nuclei  of 
four  young  amoebae. 

During  this  process  of  reproduction  the  chromatin 
at  certain  stages  of  development  occurs  as  round 
or  band-like  masses,  often  spindle-shaped,  and  several 
such  masses  may  be  scattered  through  the  cytoplasm. 
Later  these  masses  collect  into  compact  groups,  some 
of  which  take  part  in  the  formation  of  the  nuclei 
of  the  young  amoeba?  while  some  fuse  into  one  or 
two  very  compact  masses  which  remain  in  the  cyst 
as  residual  bodies. 

Conjugation. — I  have  observed  what  appears  to 
be  conjugation  in  this  species  of  amoeba,  the  process 
being  similar  to  that  described  for  Entamceba  his- 
tolytica.  The  two  conjugants  appear  to  fuse  to- 
gether and  an  interchange  of  the  cytoplasm  is  prob- 
able, as  there  are  well  marked  streaming  movements 
of  the  cytoplasm  perceptible  in  the  endoplasm  of 
both  organisms.  In  the  instances  of  this  process 
that  I  have  observed  these  phenomena  lasted  for 


190  PARASITIC  AMOEBA  OF  MAN. 

several  minutes,  after  which  the  conjugants  separated, 
but  again  became  attached  and  the  process  was  re- 
peated. After  about  half  an  hour  final  separation 
occurred,  the  organisms  moving  off  to  different 
portions  of  the  microscopic  field. 

A  similar  process  has  been  observed  by  Werner 
and  he  considers  that  one  of  the  conjugants  appears 
clearer  than  the  other  and  that  it  is  possible  to  thus 
distinguish  them. 

CULTIVATION. — Attempts  to  cultivate  Entamoeba 
tetragena  have  always  resulted  in  failure,  although 
the  most  careful  work  upon  this  subject  has  been 
done  by  Viereck,  Hartmann  and  Prowazek,  and 
Werner.  Regarding  cultivation,  Werner  says: 

"  In  no  case  did  I  succeed  in  causing  a  multiplica- 
tion of  the  vegetative  forms  of  histolytica  or  tetragena 
that  were  present  in  the  infective  matter.  But  on 
the  other  hand,  I  often  found  growth  and  encystment 
of  Amoeba  Umax,  on  the  culture  material,  and  from 
Musgrave  and  Clegg's  illustrations  I  am  convinced 
that  these  observers,  as  well  as  Walker,  who  obtained 
his  cultures  from  them,  grew  nothing  but  Amoeba 
Umax  on  their  culture  material,  and  that  it  is  this 
that  they  have  described.  It  is  certain  that  by  cats, 
and  probably  man,  the  encysted  forms  of  Amoeba 
Umax  are  often  swallowed  with  the  food,  and  traverse 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     191 

the  intestines,   to   be   excreted   in   a   condition   still 
capable  of  development." 

The  observations  of  Werner  regarding  Amoeba 
Umax  are  probably  true  as  all  the  cultivated  amoeba? 
which  I  have  observed  have  shown  a  contractile 
vacuole  which  is  not  present  in  any  parasitic  amoeba  in 
man.  The  same  negative  results  were  obtained  by 
Hartmann  and  Prowazek,  as  well  as  Werner,  in  the 
cultivation  of  Entamceba  Mstolytica,  so  that  I  believe 
that  it  is  still  doubtful  if  either  of  these  amoebae  has 
ever  been  cultivated. 

RELATION  TO  DISEASE. — The  experiments  of 
Viereck,  Hartmann  and  Prowazek,  and  Werner, 
prove  conclusively  that  Entamaeba  tetragena  pro- 
duces a  form  of  amoebic  dysentery,  but  Hartmann 
believes  that  it  is  not  as  pathogenic  for  cats  as  Enta- 
mceba Mstolytica.  The  incubation  period  in  his  ex- 
periments varied  between  8  and  10  days,  and  the  in- 
fection lasted  from  three  weeks  to  a  month.  Upon 
autopsy  the  cats  presented  typical  lesions  of  amoebic 
dysentery  and  the  amoeba  was  found  in  sections  of 
the  intestine. 

Werner,  at  the  Sailors'  Hospital  in  Hamburg, 
worked  with  five  strains  of  Entamceba  tetragena, 
only  three  of  which  he  found  to  be  pathogenic.  One 
of  these  was  still  infective  after  five,  one  after  three, 


192  PARASITIC  AMGEBJE  OF  MAN. 

and  one  after  one  passage  through  cats,  but  they  all 
lost  their  virulence  after  repeated  passage.  The  in- 
cubation period  in  these  animals  varied  from  5  to  12 
days,  the  average  being  7^  days.  The  duration 
of  the  disease  varied  from  8  to  25  days,  the 
average  being  17  days.  He  did  not  find  any  marked 
differences  between  the  lesions  produced  by  tetragena 
and  Mstolytica  and  he  does  not  believe  that  the  evi- 
dence supports  the  idea  that  tetragena  is  less  patho- 
genic than  histolytica. 

In  one  case  Werner  observed  abscess  of  the  liver 
in  a  cat  following  infection  with  Entamceba  tetragena. 
Regarding  this  case  he  says: 

"  A  cat  that  had  been  infected  per  rectum  with 
a  strain  of  tetragena  from  the  Far  East  sickened 
after  five  days'  incubation,  and  had  dysentery  symp- 
toms, amoeba*  being  found  in  the  stools,  which  con- 
tained blood  and  mucus.  After  being  ill  for  12 
days  it  died,  and  in  the  lower  portion  of  the  colon 
were  found  ulcers.  In  the  right  lobe  of  the  liver 
near  the  anterior  surface  was  found  an  abscess  as 
large  as  a  hazel-nut.  This  contained  sticky  pus,  in 
which  amoebaB  of  the  tetragena  type  were  found." 

I  have  had  no  personal  experience  with  the  ex- 
perimental production  of  dysentery  in  cats  with  Enta- 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     193 

mceba  tetragena,  but  I  consider  the  evidence  sufficient 
to  prove  that  this  species  is  capable  of  causing  dysen- 
tery in  susceptible  animals. 

THE  DIFFERENTIAL  DIAGNOSIS  OF  ENTAMCEBA 
COLL,  ENTAMCEBA  HISTOLYTICA,,  AND  ENTAMCEBA 
TETRAGENA. — As  these  species  are  most  commonly 
found  in  the  human  intestine  it  is  important  that  one 
be  able  to  distinguish  between  them,  especially  be- 
tween the  pathogenic  Entamaeba  histolytica  and 
tetragena  and  the  harmless  Entamoeba  coli. 

The  differentiation  of  these  parasites  rests  upon 
the  study  of  their  morphology  and  of  their  methods 
of  reproduction.  From  a  practical  standpoint  the 
diagnosis  must  be  made  from  the  differences  in  the 
appearance  of  the  three  species  as  they  are  observed 
in  the  feces,  and  such  a  diagnosis  can  be  made,  pro- 
vided one  cares  to  spend  the  time  necessary  for  this 
purpose.  I  do  not  mean  to  infer  that  one  who  has 
never  studied  amoeba?  can  differentiate  between 
species,  but  I  firmly  believe  that  anyone  who  has 
thoroughly  studied  these  organisms  will  be  able  to 
distinguish  species  if  sufficient  material  is  available. 
One  has  to  have  studied  free-living  forms  of  amoebae, 
as  well  as  the  forms  occurring  in  the  feces  of  man  and 
other  animals,  to  be  able  to  easily  differentiate  species 
and  much  of  the  confusion  and  inaccuracy  which  has 
arisen  in  the  classification  and  description  of  the  para- 


194  PARASITIC  AMCEBJE  OF  MAN. 

sitic  amoebae  of  man  has  been  entirely  due  to  the 
ignorance  of  investigators  regarding  the  biology  of 
this  class  of  protozoa.  It  cannot  be  denied  that  many 
observers  have  mistaken  ordinary  water  amoebae  for 
parasitic  species  and  thus  the  literature  is  filled  with 
contradictory  statements  and  absurd  deductions. 

The  differentiation  of  the  parasitic  amoebae  of  man, 
while  it  requires  experience,  is  not  very  difficult  in 
most  instances,  and  certainly  justifies  Schaudinn's 
classification.  In  a  previous  communication  I  said: 

"I  am  convinced  that  many  cases  have  been 
diagnosed  amoebic  dysentery,  which  in  reality 
presented  the  harmless  Entamceba  coli  in  the  feces, 
this  organism  being  mistaken  for  Entamoeba  histo- 
lytica.  This  mistake  might  easily  be  made  in  patients 
suffering  from  acute  enteritis,  in  which  it  is  more 
than  probable  that  the  majority  would  present  Enta- 
mceba  coli  in  the  feces,  and  this  fact  undoubtedly 
explains  the  numerous  instances  of  so-called  amoebic 
dysentery  with  rapid  and  complete  recovery. 

"  From  my  experience  there  is  no  disease  so  re- 
sistent  to  treatment  and  in  which  a  prognosis  is  so 
discouraging  as  amoebic  dysentery.  Everyone  is 
familiar  with  the  fact  that  amoebic  dysentery  recurs 
even  after  long  periods  of  time,  and  it  is  very  im- 
portant, both  to  the  patient  and  the  physician,  to 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     195 

know  absolutely  that  the  disease  being  treated  as 
amoebic  dysentery  is  in  reality  due  to  Entamceba 
histolytica,  and  that  Entamceba  coli  has  not  been 
mistaken  for  this  organism." 

Since  writing  the  above  I  have  seen  no  reason  for 
changing  my  opinion  regarding  the  importance  of 
differentiating  the  harmless  from  the  pathogenic 
amoebae,  and  I  am  convinced  that  scores  of  patients 
have  been  treated  weeks  and  even  months  for  amoebic 
dysentery  when  the  only  organism  present  was  Enta- 
mceba  coli. 

It  should  be  remembered  that  a  differential  diag- 
nosis of  these  species  does  not  rest  upon  the  presence 
of  a  single  morphological  feature  but  should  only  be 
made  after  a  careful  consideration  of  all  morpho- 
logical data  as  well  as  the  life  cycle  of  the  organisms 
investigated.  To  illustrate:  not  every  large,  motile 
amoeba,  without  a  distinct  nucleus,  is  Entamceba  his- 
tolytica,  but  if  to  these  characteristics  be  added  very 
marked  motility  and  a  clearly  differentiated  and 
highly  refractile  ectoplasm,  we  may  rest  assured  that 
we  are  dealing  with  Entamceba  histolytica  and  not 
Entamceba  coli.  Bearing  in  mind,  then,  that  our 
differential  diagnosis  must  depend  upon  the  presence 
of  several  morphological  features  rather  than  one,  the 
following  may  be  said  to  be  the  chief  points  in  which 


196  PARASITIC  AMOEBAE  OF  MAN. 

these  species  differ  from  one  another,  as  observed 
in  fresh  preparations. 

Cytoplasm. — The  very  marked  distinction  be- 
tween the  ectoplasm  and  the  endoplasm  in  Entamoeba 
histolytica  and  Entamoeba  tetragena  is  one  of  the 
most  important  features  differentiating  them  from 
Entamoeba  coli.  This  distinction  can  always  be  made 
in  the  motile  organisms,  and  very  frequently,  in  the 
case  of  Entamoeba  histolytica,  when  it  is  motionless. 
The  ecto-  and  endoplasm  in  Entamoeba  coli  can 
hardly  be  distinguished  even  when  the  organism  is 
moving  and  never  present  the  glass-like  appearance 
observed  in  the  other  species. 

Nucleus. — In  Entamoeba  coli  the  nucleus  is 
almost  always  visible,  situated  near  the  centre  of  the 
organism,  and  containing  much  chromatin,  while  it  is 
bounded  by  a  very  thick,  well  defined  nuclear  mem- 
brane. In  Entamoeba  histolytica  the  nucleus  is  gen- 
erally invisible,  and  when  visible,  is  situated  near  the 
periphery  of  the  organism;  contains  but  little 
chromatin;  and  has  no  definite  nuclear  membrane. 
In  Entamoeba  tetragena  the  nucleus  is  generally 
visible;  is  large;  contains  much  chromatin;  and  has 
.a  well  defined  nuclear  membrane.  It  is  distinguished 
from  the  nucleus  of  Entamoeba  coli  by  the  broad 
liyaline  area  surrounding  the  centriole  and  by  the 
cyclical  changes  occurring  in  the  karyosome  during 
reproduction. 


AMCEB^E  OF  THE  INTESTINAL  TRACT.     197 

Vacuoles  and  Contained  Bodies. — In  Entamceba 
histolytica  a  vacuole  is  always  present,  except  in  the 
smallest  individuals,  and  generally  there  is  more  than 
one.  This  species  also  contains  small  oval  bodies 
representing  the  nuclei  of  the  young  spores,  and  gen- 
erally one  or  more  red  blood  cells  in  cases  where  the 
feces  contain  blood.  In  Entamceba  coli  a  vacuole  is 
generally  absent  and  more  than  one  of  rare  occur- 
rence, while  the  oval  bodies  and  red  blood  cells  are 
not  observed.  In  Entamceba  tetragena  the  vacuole 
is  frequently  absent,  although  these  bodies  are  more 
often  present  in  this  species  than  in  Entamceba  coU. 
This  species  may  also  contain  red  blood  corpuscles 
when  they  are  present  in  the  feces. 

Motility. — In  both  Entamceba  histolytica  and 
Entamceba  tetragena  motility  is  very  marked,  the 
organisms  progressing  quite  rapidly  in  a  more  or 
less  definite  direction.  Under  the  same  circumstances 
Entamceba  coU  is  very  sluggishly  motile,  while  it  is 
almost  never  seen  to  progress  in  a  definite  direction. 
This  feature  alone  will  serve  to  distinguish  coli  from 
the  pathogenic  amoebae  if  the  organisms  are  observed 
in  freshly  voided  feces,  for  it  will  invariably  be  found 
that  amoebae  exhibiting  marked  motility  will  present 
morphological  features  proving  that  they  belong  to 
the  pathogenic  species. 

To  recapitulate:    if  in  a  freshly  voided  specimen 


198  PARASITIC  AMCEB^E  OF  MAN. 

of  f eces  we  observe  amoebae  showing  sluggish  motility, 
no  distinction  between  the  ecto-  and  endoplasm,  or  a 
very  slight  distinction,  and  the  presence  of  a  nucleus 
having  a  well  defined  nuclear  membrane  and  con- 
taining much  chromatin,  we  may  diagnose  the 
organism  as  Entamceba  coli;  under  the  same  con- 
ditions, if  we  observe  an  amoeba  which  is  actively 
motile,  presents  a  clear,  glass-like  ectoplasm  sharply 
distinguished  from  the  endoplasm,  and  a  nucleus  hav- 
ing a  well  defined  nuclear  membrane  and  a  clear  area 
surrounding  the  centriole,  the  diagnosis  will  be  Enta- 
mceba  tetragena;  finally,  if  an  actively  motile  amoeba 
is  observed,  showing  a  clear  distinction  between  the 
ecto-  and  endoplasm  (the  former  being  clear  and 
glass-like  in  appearance)  while  a  nucleus  is  absent, 
or  if  present,  shows  no  nuclear  membrane  and  but 
little  chromatin,  the  diagnosis  will  be  Entamceba  his- 
tolytica. 

Stained  Preparations. — In  well  stained  prepara- 
tions with  Wright's  stain  the  three  species  may  be 
distinguished  by  the  staining  of  the  ecto-  and  endo- 
plasm. In  histolytica  the  ectoplasm  stains  more  in- 
tensely than  does  the  endoplasm,  while  the  opposite 
is  true  in  Entamceba  coli.  In  Entamceba  tetragena 
there  is  but  little  distinction  between  the  staining 
reaction  of  the  ecto-  and  endoplasm.  In  stained 
specimens  showing  the  reproductive  forms  the  various 


FIG.  XXV. —  Several   vegetative  forms  of  Entamoeba   tetragena,  some  of 
which  contain  numerous  red  blood-corpuscles.     (After  Viereck.) 


FIG.  XXVI. — Encysted  forms  of  Amoeba  Umax,  which  are  often  mistaken 
for  those  of  Entamoeba  histolytica  and  Entamoeba  coli. 


AMOSBJ5  OF  THE  INTESTINAL  TRACT.     199 

species  may  be  distinguished  by  the  amount  and 
arrangement  of  the  nuclear  chromatin,  as  has  already 
been  described. 

METHOD  OF  REPRODUCTION. — To  one  who  cares  to 
devote  the  time  and  study  necessary,  the  investiga- 
tion of  the  methods  of  reproduction  of  the  species 
under  discussion  will  definitely  distinguish  them. 
While  they  all  reproduce  by  simple  division  when 
conditions  are  favorable  to  vegetative  existence,  they 
differ  widely  in  their  methods  of  reproduction  under 
other  conditions.  Entamceba  histolytica  reproduces 
by  spore  formation,  the  young  spores  being  budded 
off  from  the  periphery  of  the  mother  organism,  a 
method  totally  different  from  that  of  Entamceba  coU 
which  encysts,  eight  daughter  amoebge  being  formed 
within  the  cyst.  Under  the  same  conditions  Enta- 
mceba  tetragena  undergoes  encystment,  but  only  four 
daughter  amoebse  are  formed  within  the  cyst.  At  a 
certain  stage  in  the  development  of  Entamceba  coli 
the  cyst  contains  only  four  nuclei  and  care  must  be 
taken  not  to  mistake  this  stage  for  the  cysts  of  Enta- 
mceba tetragena.  These  differences  in  the  reproduc- 
tive cycle  are  of  the  greatest  importance  in  the  differ- 
entiation of  species  and  whenever  there  is  any  doubt 
concerning  the  nature  of  an  amoeba  present  in  a  given 
case  the  methods  of  reproduction  should  always  be 
studied. 


200  PARASITIC  AMCEBJE  OF  MAN. 

In  order  to  facilitate  the  diagnosis  of  the  three 
species  under  discussion  I  have  prepared  Table  IV, 
giving  the  chief  differential  points  between  them. 

ENTAMCEBA   MI  NUT  A.     Elmassian,  1909. 

Elmassian  has  described  an  amoeba  in  cases  of 
dysentery  occurring  in  Paraguay  which  he  considers 
a  new  species  and  to  which  he  has  given  the  name 
Entamceba  minuta  because  of  its  small  size,  specimens 
seldom  exceeding  14  microns  in  diameter. 

GEOGRAPHICAL  DISTRIBUTION. — So  far  as  is 
known  this  species  occurs  only  in  South  America, 
and  at  the  present  writing  has  been  described  as 
occurring  in  Paraguay,  but  will  probably  be  found 
distributed  throughout  South  America,  and  the  ad- 
jacent islands. 

MORPHOLOGY. — Entamceba  minuta  closely  re- 
sembles morphologically  both  Entamceba  histolytica 
and  Entamceba  tetragena,  but  is  distinguished  from 
them  by  its  small  size.  From  Elmassian's  descrip- 
tion it  would  appear  that  this  feature  is  of  the  greatest 
importance  in  the  differentiation  of  the  species  and 
if  his  observations  are  confirmed  the  question  of 
the  size  of  an  amoeba,  hitherto  considered  as  of  slight 
specific  importance,  will  prove  to  be,  in  this  instance, 
at  least,  a  valuable  distinguishing  feature.  The  de- 
scription which  follows  is  compiled  from  Elmassian's 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     201 

paper,  as  I  have  had  no  personal  experience  with  this 
species  of  amoeba. 

Size. — Elmassian  states  that  the  small  size  of  this 
organism  is  distinctive.  The  average  measurement 
is  12  to  14  microns  in  diameter,  but  sometimes  the 
organism  may  measure  as  much  as  16  or  18  microns. 
In  one  instance  Elmassian  observed  an  amoeba 
measuring  20  microns  in  diameter,  but  he  considered 
that  it  probably  belonged  to  another  species. 

Shape. — When  motionless  this  parasite  is  spher- 
ical, or  slightly  oval  in  shape,  but  when  moving  many 
variations  occur  in  the  shape  of  the  organism  due  to 
the  extrusion  of  pseudopodia. 

The  Protoplasm. — The  protoplasm  consists  of  an 
alveolar  structure  containing  a  nucleus  which  is  situ- 
ated near  the  centre  of  the  organism.  A  vacuole  is 
often  present  and,  when  blood  is  present  in  the  feces, 
the  amoebae  frequently  contain  red  blood  corpuscles. 

The  Cytoplasm. — The  cytoplasm  of  Entamceba 
minuta  is  divided  into  a  well  marked  ecto-  and  endo- 
plasm,  but  these  two  portions  cannot  be  distinguished 
unless  the  organism  is  moving.  The  ectoplasm  is 
clear  and  very  refractive  in  appearance,  while  the 
endoplasm  is  granular  in  structure  and  grayish  in 
color.  Under  high  power  the  endoplasm  appears 
to  be  composed  of  multitudes  of  granules  arranged 
in  an  alveolar  ground  substance. 


202 


PARASITIC  AMOEBAE  OF  MAN. 


TABLE 
DIFFERENTIAL  FEATURES  OF  ENTAMGEBA  COLI, 


Name. 

Size. 

Pseudopodia. 

Motility. 

Protoplasm. 

Entamaeba  coli 

10  to  30  microns 

Small  blunt  and 

Ectoplasm   not   distinct 

Schaudinn,  1903. 

Generally  smal- 
ler than   Enta- 
moeba  histolyt- 
ica     or     Enta- 
mceba tetragena. 

not  clearly  dif- 
ferentiated from 
rest  of  parasite. 

except  when  moving 
and  then  only  because 
it  is  free  from  granules. 
Is  grayish  in  color  and 
not  very  refractive. 
Endoplasm  is  gray,  fine- 
ly granular,  few  non- 
contractile  vacuoles.  Is 
not  generally  phagocytic 
for  red  blood  corpuscles. 

Entamceba  histolytica  

10  to  70  microns. 

Blunt  or  slender 

Active 

Ectoplasm    is    very    dis- 

Schaudinn, 1903. 

Generally  from 
15    to    40    mi- 
crons. 

and  finger-shap- 

fractive  and 
clearly  differen- 
tiated from  rest 
of  the  parasite. 

tinct  and  refractive,  in 
some  instances  even 
when  motionless.  Glassy 
appearing. 

Endoplasm  is  granular, 
contains  numerous  non- 
contractile  vacuoles  and 
red  blood  corpuscles 
when  latter  are  present 
in  the  feces. 

Entamceba  tetragena  

10  to  50  microns. 

Viereck,  1907. 

About  the  size 
of    Entamceba 
histolytica. 

shaped.  Very 
refractive  and 
well  differenti- 
ated from  rest 
of  parasite. 

plasm  well  differentiated  . 
Ectoplasm  hyaline  in 
appearance.  Endoplasm 
granular,  containing  nu- 
merous non-contractile 
vacuoles  and  red  blood 
corpuscles  when  latter 
are  present  in  the  feces. 

AMGEBJE  OF  THE  INTESTINAL  TRACT.     203 


IV. 

ENTAMCEBA  HISTOLYTICA,  AND  ENTAMCEBA  TETRAGENA. 


Nucleus. 

Cyst 
Formation. 

Cultiva- 
tion. 

Methods  of 
Reproduction  . 

Pathogenesis. 

Staining. 

Distinct,  having    Present.    Eight 

Doubtful 

By  simple  division, 

Is    not    patho- 

With   Wright's 

a  well  denned 
nuclear  mem- 
brane  and 
much  chroma- 

young  amoebae 
developed 
within  cyst. 

autogamous    sex- 
ual    reproduction 
in    cyst,    and    by 
schizogony     with 

genic,     occur- 
ring in  a  large 
percentage   of 
healthy    indi- 

stain,   ecto- 
Elasm    light 
lue,    endo- 
plasm    dark 

tin. 

the  production  of 

viduals,  and  in 

blue,  and  chro- 

Large k  a  r  y  o  - 

eight        daughter 
amoebse.        Eight 

patients     suf- 
fering   from 

matin    of    nu- 
cleus, red. 

some. 

amoebae    are   pro- 

diseases other 

duced  within  the 

than  d  y  s  e  n  - 

cyst. 

tery. 

Indistinct.     No 
well   defined 
nuclear  mem- 
brane and  but 

Minute  spores 
developed  by 
budding  from 
the  parent  or- 

Doubtful 

By  simple  division, 
gemmation,    and 
by  the  budding  of 
chromidial  masses 

Is  the  qau.se  of  a 
form  of  amoe- 
bic dysentery. 

With     Wright's 
stain,   e  c  t  o  - 
plasm    dark 
blue,   e  n  d  o  - 

little  chroma- 

ganism.  Meas- 

surrounded  by 

plasm    light 

tin.       Minute 

ure  from  3  to  5 

protoplasm     from 

blue,  and  chro- 

karyosome. 

microns.  Are 

the    periphery    of 

matin    of    the 

covered  with  a 
:     c  y  s  t  -  1  i  k  e 

the  mother  para- 
site,  forming   the 

nucleus      pale 
red  or  pink. 

membrane. 

so-called  cystic 

spores., 

Distinct,  having 
definite    n  u  - 

Present.  Four 
amoebae  devel- 

Negati v^e 

By  simple  division, 
and  by  autogam- 

Is the  cause  of  a 
form  of  amoe- 

Does   not    stain 
well   with 

clear       mem- 
brane  formed 

op  within  cyst. 

ous  sexual  repro- 
d  u  c  t  i  o  n    within 

bic  dysentery. 

Wright's  stain. 
Chromatin 

by  chromatin. 

cyst,  four  amoebae 

takes  a  bright 

Large    karyo- 

being produced. 

red  stain. 

some.       Clear 

area  surround- 

ing   the    cen- 

triole. 

204  PARASITIC  AMOEBAE  OF  MAN. 

The  Nucleus. — This  structure  is  always  well  de- 
fined and  is  remarkable  for  its  richness  in  chromatin 
and  for  its  regularly  spherical  form.  A  minute  karyo- 
some  can  be  distinguished  containing  a  collection  of 
fine  granules  which  are  situated  at  the  centre,  form- 
ing a  centriole.  In  the  vegetative  stage  the  diameter 
of  the  nucleus  does  not  exceed  2^  to  3  microns,  but 
in  the  cystic  stage  the  diameter  of  the  nucleus  is  from 
4  to  6  microns.  The  chromatin  is  collected  at  the 
periphery  of  the  nucleus,  thus  giving  rise  to  a  very 
solid,  refractive  nuclear  membrane.  During  repro- 
duction the  chromatin  is  scattered  throughout  the 
nuclear  substance  and  becomes  arranged  in  threads 
and  granules.  In  some  instances  a  wreath  of 
chromatin  granules  may  be  observed  surrounding  the 
minute  centriole. 

Vacuoles  and  Contained  Bodies. — A  contractile 
vacuole  has  not  been  observed  in  this  species,  and 
very  frequently  vacuoles  are  absent,  even  in  well 
developed  organisms.  A  single  vacuole  is  most  fre- 
quently observed,  but  there  may  be  two  or  three, 
especially  in  organisms  undergoing  division.  This 
species  is  phagocytic  for  red  corpuscles  and  also  for 
certain  parasites  occurring  in  the  feces.  Elmassian 
states  that  he  has  observed  Chlamydophrys  stercorea 
within  amoebae  of  this  species.  Like  other  amoebae, 
this  organism  often  contains  bacteria  and  crystals  of 
various  kinds. 


OF  THE  INTESTINAL  TRACT.     205 

Motility. — Entamceba  minuta  is  actively  motile, 
the  pseudopodia  being  composed  entirely  of  ecto- 
plasm. It  is  only  in  the  moving  organism  that  the 
ectoplasm  can  be  distinguished  and  it  resembles  that 
of  histolytica  and  tetragena,  being  clear  and  glass- 
like  in  appearance.  The  pseudopodia  are  generally 
short  and  blunt,  but  may  sometimes  be  finger-shaped, 
especially  in  the  more  actively  motile  amoebae. 

Reproduction. — The  reproductive  processes  in 
this  species  are  very  similar  to  those  occurring  in 
Entamceba  tetragena,  consisting  of  simple  division, 
schizogony,  and  reproduction  within  a  cyst. 

Simple  division  is  preceded  by  the  mitotic  division 
of  the  nucleus  after  which  the  cytoplasm  divides, 
two  amcebge  being  produced.  The  process  is  similar 
to  that  in  other  amoebse,  and  has  been  described  in 
preceding  sections. 

Schizogony  results  in  the  division  of  the  organism 
into  four  young  amoeba?.  It  is  initiated  by  the 
division  of  the  nucleus  by  mitosis  into  four  portions 
and  is  completed  by  the  division  of  the  cytoplasm. 
During  the  division  of  the  nucleus  a  well  marked 
equatorial  plate  is  frequently  observed. 

Reproduction  within  a  cyst  is  initiated  by  the  ex- 
trusion from  the  organism  of  all  foreign  particles, 
the  cytoplasm  becoming  clear  in  appearance,  while 
the  nucleus  increases  in  size  to  twice  its  former 


206  PARASITIC  AMCEBJE  OF  MAN. 

diameter.  The  chromatin  becomes  arranged  in  semi- 
lunar  bands  crossing  the  nucleus  and  eventually  an 
equatorial  plate  is  formed,  after  which  the  nucleus 
divides  into  two  equal  portions.  The  nuclei  now 
undergo  similar  changes  to  those  described  in  auto- 
gamie  in  Entamceba  coli  and  finally  four  nuclei  are 
produced  representing  the  nuclei  of  the  four  daughter 
amoebae. 

The  cysts  are  generally  spherical  in  shape  and 
measure  12  microns  in  diameter,  although  some  are 
observed  as  small  as  10  microns  and  as  large  as  14 
microns.  During  the  process  of  encystment  a 
gelatinous  membrane  is  formed  having  a  double  out- 
line. The  protoplasm  of  the  cyst  at  certain  stages 
of  development  appears  reticular,  the  alveoli  being 
minute  in  size.  Sometimes  cysts  are  observed  con- 
taining three  nuclei,  but  Elmassian  considers  that 
these  are  degenerating  organisms. 

Relation  to  Disease. — Elmassian  does  not  give 
any  data  regarding  the  experimental  production  of 
dysentery  in  animals  with  material  containing  this 
species  of  amoebse,  basing  his  belief  in  the  pathogenic 
nature  of  the  organism  upon  its  constant  occurrence 
in  the  feces  of  patients  suffering  from  diarrhoea  and 
dysentery.  From  his  description  it  is  apparent  that 
this  parasite  bears  a  very  close  resemblance  in  mor- 
phology and  methods  of  reproduction  to  Entamceba 


FIG.  XXVII. — Various  stages  in  the  life  history  of  Entamoeba  minuta. 
(After  Elmassian.)  1,  vegetative  forms  which  are  motile,  showing  the  dis- 
tinction between  the  ecto-  and  endoplasm  and  the  lack  of  a  visible  nucleus. 
Note  the  markedly  lobose  pseudopodium;  2  and  5,  Chlamydophrys  ster- 
coria,  a  parasite  which  is  often  found  within  Entamoeba  minuta;  3,  motile 
and  immotile  forms  of  Entamceba  minuta.  Note  lack  of  distinction  between 
the  ecto-  and  endoplasm  in  the  motionless  forms;  4,  young  amoebae  pro- 
duced by  schizogony;  6,  vegetative  form  containing  a  parasite;  7,  vegetative 
form  of  Entamoeba  coli;  8,  encysted  amoebae  containing  spore-like  bodies; 
9,  encysted  Entamoeba  minuta,  showing  the  appearance  of  the  nucleus  prior 
to  division;  10,  encysted  amoebae  containing  two  other  parasites  which  con- 
tain spores;  11  and  12,  encysted  amoebae,  showing  division  of  the  nucleus; 
13,  encysted  Entamoeba  coli,  showing  division  of  the  nucleus  into  four  parts, 
one  of  which  shows  mitosis;  14,  15,  and  16,  encysted  Entamceba  minuta, 
showing  division  of  the  nucleus  into  four  daughter-nuclei;  17  and  18,  encysted 
Entamoeba  coli,  showing  in  17  the  four  nuclear  stage,  two  of  the  nuclei 
undergoing  mitosis,  and  in  18  the  eight  nuclear  stage  of  this  parasite. 


FIG.  XXVII 


AMCEBJE  OF  THE  INTESTINAL  TRACT.     207 

tetragena  and  the  fact  that  the  latter  species  also 
occurs  in  South  America  throws  some  doubt  upon 
the  validity  of  the  species  described  by  this  author. 
The  one  point  of  distinction  appears  to  be  the  uni- 
formly minute  size  of  Entamceba  minuta  and  if 
further  observation  confirms  the  statements  of 
Elmassian  in  this  respect  it  may  possibly  be  accepted 
as  a  new  species,  but  it  should  be  remembered  that 
mere  difference  in  size  must  always  be  a  doubtful 
feature  upon  which  to  base  a  specific  distinction. 

ENTAMCEBA  NIPPONICA.    Koidzumi,  1909. 

This  species  of  amoeba  was  described  in  1909  by 
Koidzumi.  He  observed  it  first  in  an  advanced  case 
of  dysentery  in  a  Japanese  in  conjunction  with  Enta- 
moeba  histotytica,  but  later  he  found  it  present  in 
mild  cases  of  the  amoebic  type  of  dysentery,  and  in 
some  cases  of  bacillary  dysentery.  He  also  states 
that  it  is  not  infrequently  observed  in  patients  suffer- 
ing from  diarrhoea. 

GEOGRAPHICAL  DISTRIBUTION. — The  geographical 
distribution  of  Entamceba  nipponica  is  confined  to 
Japan  so  far  as  is  at  present  known,  but  if  this  be 
a  distinct  species  it  is  probable  that  it  will  be  found 
widely  distributed  in  the  Far  East. 

MORPHOLOGY. — In  the  vegetative  stage  of  devel- 
opment this  amoeba  possesses  the  general  features  of 
the  Umax  group,  but  the  pseudopodia  are  never 


208  PARASITIC  AMOEBA  OF  MAN. 

spinose.  A  distinct  difference  exists  in  the  appear- 
ance of  the  ecto-  and  endoplasm,  while  it  reproduces 
by  simple  division,  schizogony,  and  by  spore  forma- 
tion within  a  cyst. 

Size. — Koidzumi  gives  the  average  size  of  this 
amoeba  as  25  microns  in  its  longest  diameter,  but 
it  varies  from  20  to  30  microns  in  length  and  15  to 
20  microns  in  breadth.  It  seldom  measures  over 
40  microns  in  its  longest  diameter.  There  is  nothing 
characteristic  about  the  size  of  this  organism,  except 
that  it  is  longer  in  one  diameter  than  in  the  other. 

Shape. — When  motionless  Entamceba  wpponica 
is  always  oval  in  shape,  according  to  Koidzumi,  which 
differentiates  it  from  the  other  species  of  amoebae 
which  have  been  described,  all  of  which  are  spherical 
in  shape  when  motionless.  Numerous  changes  occur 
in  the  shape  of  the  organism  during  motility  due  to 
the  extrusion  of  pseudopodia. 

The  Cytoplasm. — The  cytoplasm  of  this  species 
much  resembles  that  of  Entamceba  tetragena,  being 
divided  into  a  well  marked  ecto-  and  endoplasm. 
The  ectoplasm,  however,  forms  but  a  very  small 
proportion  of  the  cytoplasm,  existing  as  a  narrow 
rim  surrounding  the  endoplasm.  When  moving  the 
ectoplasm  appears  larger  in  amount  because  it  is 
concentrated  in  the  pseudopodia.  It  is  clear,  color- 
less and  very  refractive.  The  endoplasm,  comprising 


AMCEB.E  OF  THE  INTESTINAL  TRACT.     209 

about  seven-eighths  of  the  organism,  is  light  gray  in 
color,  very  refractive,  and  granular  in  structure,  the 
granules  being  much  coarser  than  in  Entamceba  his- 
tolytica  or  in  Entamceba  tetragena.  The  endoplasm 
always  contains  non-contractile  vacuoles  of  various 
size. 

The  Nucleus. — The  nucleus  is  always  visible  as  a 
well  defined  spherical  body,  measuring  from  5  to  7 
microns  in  diameter,  and  situated  near  the  centre  of 
the  parasite.  A  very  delicate  but  distinct  nuclear 
membrane  surrounds  the  nucleus.  The  chromatin  is 
large  in  amount  and  occurs  in  masses  upon  the  inner 
surface  of  the  nuclear  membrane,  there  being  no 
chromatin  whatever  distributed  through  the  nuclear 
plasma.  During  the  vegetative  stage  the  chromatin 
appears  as  refractive  masses  varying  in  shape  which 
lie  upon,  or  are  attached  to,  the  nuclear  membrane. 
Sometimes  they  are  crescent  or  spindle  shaped  and 
lie  lengthwise  upon  this  membrane  or  they  may  be 
attached  to  it  by  delicate  fibrils.  The  masses  of 
chromatin  vary  in  number  from  3  to  8,  according 
to  the  age  of  the  parasite,  being  more  numerous  in 
the  older  organisms. 

Vacuoles  and  Contained  Bodies. — A  contractile 
vacuole  is  not  present  in  this  species,  but  the  endo- 
plasm always  contains  one  or  more  non-contractile 
vacuoles  which  vary  in  size.  In  cases  where  the  feces 


14 


210  PARASITIC  AMOEBA  OF  MAN. 

contain  blood  erythrocytes  may  be  observed  within 
the  endoplasm,  as  this  parasite  is  phagocytic  for  these 
cells.  Bacteria  and  other  crystals  occur  within  the 
endoplasm  as  in  other  amoebae. 

Motility. — This  species  is  actively  motile,  the 
pseudopodia  being  short  and  blunt.  Koidzumi  states 
that  the  pseudopodia  do  not  change  their  shape  dur- 
ing motion. 

Reproduction. — Entamceba  nipponica  reproduces 
by  simple  division,  schizogony,  and  by  the  formation 
of  daughter  amoebae  within  a  cyst.  In  reproduction 
by  simple  division,  Koidzumi  states  that  the  nucleus 
divides  amitotically  into  two  portions,  followed  by  the 
division  of  the  cytoplasm.  Simple  division  has  been 
observed  even  in  the  very  young  amoebae. 

Schizogony  is  initiated  by  the  crescent  or  spindle 
shaped  masses  of  chromatin  changing  to  spheres  of 
nearly  equal  size,  which  vary  in  number  from  six  to 
eight,  and  remain  attached  to  the  nuclear  membrane 
by  a  delicate  filament.  The  nuclear  membrane  gradu- 
ally disappears  and  the  chromatin  masses  become 
free  in  the  cytoplasm.  Each  mass  is  then  surrounded 
by  a  portion  of  the  latter  and  the  entire  organism 
breaks  up  into  as  many  young  amoebae  as  there  are 
masses  of  chromatin.  The  young  amrebae  are  oval 
in  shape  and  measure  about  5  microns  in  diameter. 
The  cytoplasm  is  dense  in  appearance  and  stains 


AMQEBJE  OF  THE  INTESTINAL  TRACT. 

deeply.  The  nucleus  consists  at  first  of  a  single 
mass  of  chromatin,  but  later  a  nuclear  membrane  is 
developed.  The  chromatin  assumes  a  crescentic  .shape 
and  separates  into  two  portions  which  eventually 
break  up  into  several  small  masses  situated  upon  the 
inner  side  of  the  nuclear  membrane.  While  these 
changes  are  occurring  in  the  nucleus  the  cytoplasm 
becomes  differentiated  into  an  ecto-  and  endoplasm, 
vacuoles  appear,  and  the  organism  gradually  assumes 
the  appearance  typical  of  the  fully  developed  vege- 
tative stage. 

Encystment  is  first  indicated  by  a  reduction  in 
the  size  of  the  parasite,  although  the  oval  shape  is 
retained.  A  delicate  cyst  wall  is  gradually  developed 
having  a  single  outline.  Some  of  the  chromatin 
masses  of  the  nucleus  become  spherical  in  shape  and 
pass  through  the  nuclear  membrane  into  the  cyto- 
plasm, where  they  remain  unchanged,  so  far  as  he 
was  able  to  observe.  The  chromatin  remaining  in 
the  nucleus  divides  into  minute  granules  which  be- 
come evenly  distributed  over  the  inner  surface  of  the 
nuclear  membrane.  Koidzumi  was  unable  to  follow 
the  further  development,  so  that  we  are  in  ignorance 
of  the  further  changes  occurring  in  the  nucleus  and 
of  the  number  of  daughter  amoeba?  which  are 
produced  within  the  cyst. 

RELATION  TO  DISEASE. — The  author  gives  no  data 


PARASITIC  AMOEBAE  OF  MAN. 

regarding  experiments  upon  animals  with  this  species 
of  amoeba.  He  believes  that  it  is  the  cause  of  a  mild 
form  of  dysentery  in  Japan  and  bases  his  belief  upon 
the  occurrence  of  the  organism  in  the  feces  of  pa- 
tients suffering  from  this  type  of  the  disease. 

As  regards  the  validity  of  this  species  it  must 
be  admitted  that  at  this  time  the  data  are  hardly 
sufficient  to  definitely  prove  that  the  parasite  de- 
scribed by  Koidzumi  is  a  distinct  species.  Its  re- 
semblance, according  to  his  description,  to  both 
Entamoeba  histolytica  and  Entamoeba  tetragena, 
leaves  one  in  doubt  as  to  whether  he  may  not  have 
mistaken  certain  stages  of  the  development  of  either 
of  these  parasites  for  a  new  species. 

.ENTAMCEBA    TROPICALIS.     Lesage,  1908. 

From  his  observations  Lesage  has  concluded  that 
the  harmless  amoeba  found  in  healthy  individuals  and 
in  those  suffering  from  diseases  other  than  dysentery 
in  the  Tropics,  belongs  to  a  distinct  species,  and  to 
this  parasite  he  has  given  the  name  Entamoeba 
tropicaUs. 

The  species  resembles  Entamoeba  coli  in  general 
appearance.  It  has  a  distinct  nucleus,  which  contains 
much  chromatin,  and  reproduces  by  simple  division 
and  by  the  development  of  daughter  amoeba?  within  a 
cyst.  It  differs  from  Entamoeba  coli  in  having  a 


AMCEByE  OF  THE  INTESTINAL  TRACT.     213 

distinct  ectoplasm  and  by  the  fact  that  it  can  be  cul- 
tivated in  symbiosis  with  bacteria  of  various  kinds. 
The  cysts  are  smaller  than  those  of  Entamoeba  coli 
and  instead  of  eight  daughter  amoebae  being  formed 
within  the  cyst  this  species  produces  from  three  to 
as  many  as  thirteen  daughter  amoebae. 

Lesage  believes  that  this  is  the  species  which  has 
been  cultivated  by  Musgrave  and  Clegg,  and  that  the 
production  of  dysentery  in  animals  with  such  cultures, 
as  reported  by  the  latter  observers,  was  due  to  con- 
tamination with  the  spores  of  Entamoeba  histotytica. 

According  to  Lesage,  Entamoeba  tropicalis  is  not 
pathogenic  for  animals. 

I  have  not  been  able  to  confirm  the  observations 
of  Lesage  regarding  the  existence  of  a  distinct  species 
of  harmless  amoeba  in  the  tropical  regions  in  which 
I  have  studied  this  subject,  and  I  believe  it  very 
doubtful  if  this  species  is  valid. 

ENTAMCEBA   PHAGOCYTOIDES.     Gauducheau,  1908. 

This  species  was  described  by  Gauducheau,  who 
found  it  in  the  feces  of  a  case  of  dysentery  occurring 
in  Indo- China.  Its  characteristic  features  are  its 
small  size,  from  two  to  fifteen  microns  in  diameter; 
the  presence  of  a  well  marked  ectoplasm;  and  the 
fact  that  it  can  be  easily  cultivated  on  ordinary  agar- 
agar  in  symbiosis  with  bacteria.  The  author  gives 


PARASITIC  AMCEBJE  OF  MAN. 

no  data  concerning  its  relation  to  dysentery,  beyond 
its  occurrence  in  a  single  case  of  this  disease.  At  the 
present  writing  his  observations  have  not  been  con- 
firmed. 

ENTAM(EBA   UNDULANS.     Castellani,  1905. 

Castellani  describes  an  organism  occurring  in  the 
feces  of  patients  in  Ceylon,  suffering  from  diarrhoea, 
which  he  considers  a  new  species  of  amoeba,  and  to 
which  he  has  given  the  name  of  Entamceba  undulans. 
The  organism  measures  from  25  to  30  microns  in 
diameter,  is  oval  or  round  in  shape  and,  unlike  other 
amoebae,  it  possesses  an  undulating  membrane  which 
is  in  constant  motion.  A  long  narrow  pseudopodium 
is  rapidly  extruded  from  the  body  of  the  parasite  at 
frequent  intervals  and  is  quickly  withdrawn,  and  only 
one  pseudopodium  is  extruded  at  a  time.  The  proto- 
plasm is  finely  granular  and  there  is  no  distinction 
between  the  ecto-  and  endoplasm.  The  nucleus  is 
generally  invisible  and  the  endoplasm  contains  a 
single  small  vacuole,  varying  in  position.  He  was 
unable  to  observe  division  and  no  encysted  forms  were 
noticed. 

The  close  resemblance  of  this  parasite  to  certain 
stages  in  the  life-cycle  of  Trichomonas  intestinalis 
suggests  that  the  author  may  have  been  observing 
forms  of  the  latter  organism,  although  he  states  that 


AMOEBA  OF  THE  INTESTINAL  TRACT.     215 

the  parasite  is  much  larger  than  Trichomonas,  and 
as  both  it  and  Entamoeba  Mstolytica  were  present  in 
the  feces  of  some  of  the  cases  examined,  comparison 
was  easy,  and  he  is  sure  that  it  did  not  correspond 
to  either  of  the  latter  organisms.  His  observations 
have  not  been  confirmed. 

PARAMCEBA   HOMINIS.     Craig,  1906. 

In  August,  1906,  I  published  the  description  of 
a  new  species  of  amoeba  to  which  I  gave  the  name 
of  Paramceba  hominis.  The  genus,  Paramceba,  was 
established  in  1896  by  Schaudinn,  to  include  a  water 
amoeba  which  he  described  at  that  time.  Schaudinn's 
species  occurred  in  sea  water  and  was  peculiar  in 
that  a  flagellate  stage  of  development  alternated  with 
an  amoebic  stage.  The  organism  described  by  him, 
after  multiplying  for  several  generations  by  simple 
division,  at  the  end  of  its  vegetative  life  becomes  en- 
cysted, and  within  the  cyst  there  develop  swarm- 
spores  which  are  liberated,  and  after  living  as  flagel- 
lates and  multiplying  by  longitudinal  division,  finally 
lose  their  flagellum  and  again  become  typical  amoeba?. 
Schaudinn  described  the  process  of  spore  formation 
as  consisting  in  the  division  of  the  nucleus  of  the 
encysted  amoeba,  this  division  being  preceded  by  the 
division  of  a  cytoplasmic  body  lying  in  contact  with 
the  nucleus,  which  acts  as  a  centrosome  or  blepharo- 


216  PARASITIC  AMOEBAE  OF  MAN. 

plast.  The  number  of  spores  corresponds  to  the 
number  of  the  divisions  of  the  centrosome,  each 
swarm-spore  consisting  of  a  portion  of  the  original 
nucleus  and  of  the  cytoplasmic  body. 

After  the  formation  of  the  swarm-spores  is  com- 
plete they  develop  a  flagellum,  escape  from  the  cyst, 
and  after  swimming  about  actively  for  an  indefinite 
time,  undergo  longitudinal  division  and  finally,  after 
losing  their  flagella,  develop  into  typical  amoeba?, 
which  multiply  by  simple  division  and  again  repeat 
the  process  of  encystment  and  spore  formation. 

Schaudinn  placed  this  organism  in  a  new  genus, 
Paramceba,  and  gave  it  the  specific  name,  eilhardi. 
Until  my  description  the  genus  was  not  known  to 
contain  any  organism  living  within  man. 

Paramceba  homirds  passes  through  both  an 
amoebic  and  a  flagellate  stage  during  its  life-cycle,  and 
for  this  reason  and  because  the  organism  morpholog- 
ically resembled  Paramceba  eilhardi,  I  had  no  hesita- 
tion in  placing  it  in  Schaudinn's  genus,  Paramceba. 
Doflein  is  inclined  to  think  that  further  research  will 
show  that  the  parasite  I  described  should  be  placed 
in  a  new  genus,  but  certainly  all  the  evidence  in- 
dicates that  it  belongs  to  the  genus  Paramceba.  The 
life-cycle  is  similar  in  every  respect  to  that  of  Para- 
moeba  eilhardi  and  I  am  therefore  convinced  that 
this  parasite  is  properly  placed  in  the  genus  Para- 


AMCEBJE  OF  THE  INTESTINAL  TRACT. 

maeba,  and  that  this  genus  must  now  be  regarded  as 
containing  a  species  capable  of  existing  as  a  parasite 
in  man. 

GEOGRAPHICAL  DISTRIBUTION. — This  species  of 
amoeba  was  first  observed  in  the  feces  of  a  Filipino 
suffering  from  an  attack  of  chronic  diarrhoea  and  the 
same  organism  was  afterwards  found  in  the  feces  of 
five  other  Filipinos.  At  the  time  that  I  published 
my  original  description,  I  had  never  observed  the 
parasite  in  Americans  or  Europeans,  although  I 
searched  very  carefully  for  it,  and  many  of  the 
Americans  examined  had  resided  in  the  Philippines 
for  considerable  periods  of  time. 

In  1908,  while  serving  at  Fort  Leavenworth,  Kan- 
sas, I  was  able  to  study  the  same  parasite  in  three 
American  soldiers  who  had  just  returned  from  the 
Philippine  Islands,  and  all  of  whom  entered  the  hos- 
pital because  of  recurring  attacks  of  diarrhoea. 

In  all  probability  these  men  became  infected  in 
the  Philippines,  and  it  may  well  be  that  this  parasite 
occurs  there  much  more  frequently  than  has  been 
supposed,  having  been  confused  with  Entamceba  his- 
tolytica,  Entamceba  tetragena,  Entamceba  coli,  or 
Trichomonas  hominis. 

So  far  as  the  evidence  goes,  the  geographical  dis- 
tribution of  this  species  appears  to  be  confined  to  the 
Philippine  Islands,  but  I  am  of  the  opinion  that  care- 


218  PARASITIC  AMGEB.E  OF  MAN. 

fill  research  will  result  in  proving  that  the  parasite 
also  occurs  in  this  country,  for  chronic  forms  of  diar- 
rhoea frequently  occur  in  many  portions  of  the  United 
States  and  monads  are  frequently  reported  as  occur- 
ring in  the  feces  of  such  cases.  It  is  very  easy  to 
confuse  the  flagellate  stage  of  Paramceba  homims 
with  monads  unless  one  is  well  acquainted  with  the 
protozoa  occurring  in  the  intestine  of  man,  and  I 
believe  that  it  is  not  at  all  unlikely  that  many  of  the 
cases  of  so-called  monadic  diarrhoea,  or  dysentery,  are 
in  reality  infections  with  Par  amoeba  hominis.  It  must 
be  admitted,  however,  that  infection  with  this  para- 
site is  rare  as  compared  with  the  other  species  of 
amoebse  infesting  man,  or  with  infections  with  Tricho- 
monas  hominis,  Lamblia  intestinaUs,  or  even  Balan- 
tidium  coli,  for  in  my  own  experience,  covering  the 
microscopical  examination  of  several  thousand  speci- 
mens of  feces  from  as  many  individuals,  both  in  health 
and  disease,  I  have  found  this  organism  in  only  nine 
patients,  three  of  whom  were  American  soldiers,  and 
six  native  Filipinos. 

MORPHOLOGY  AND  LIFE-CYCLE. — This  parasite  has 
a  complicated  life-cycle,  passing  through  both  an 
amoebic  and  a  flagellate  stage  of  existence.  By  making 
repeated  examinations  of  the  feces  of  infected  in- 
dividuals I  have  been  able  to  trace  the  entire  life- 
cycle  and  while  I  have  been  unable  to  reach  definite 


AMOEBAE  OF  THE  INTESTINAL  TRACT.     219 

conclusions  regarding  certain  points  connected  with 
the  developmental  history,  such  as  the  conditions  hast- 
ening or  retarding  the  various  stages  of  growth,  or 
the  intervals  of  time  which  elapse  between  the  amoebic 
and  the  flagellate  stage,  and  the  time  consumed  in 
the  development  of  swarm-spores,  I  have  been  able 
to  study  each  stage  of  development  and  to  confirm 
in  this  species  the  description  given  by  Schaudinn  of 
the  life-cycle  of  Paramceba  eilhardi. 

Beginning  with  the  amoebic  stage,  the  parasite 
reproduces  by  simple  division  for  a  certain  period, 
probably  for  as  long  as  conditions  are  favorable  to 
its  vegetative  existence,  and  during  this  time  its  struc- 
ture is  that  of  a  typical  amoeba.  Encystment  is  in- 
itiated by  the  organism  becoming  motionless,  assum- 
ing a  spherical  shape,  and  then  rotating  rapidly,  the 
cyst  wall  being  formed  during  the  process  of  rota- 
tion. When  encystment  is  complete,  the  organism 
again  becomes  motionless,  and  a  refractive,  double- 
outlined  cyst  wall  is  then  distinguishable.  Within 
the  cyst  there  soon  appear  numerous  small,  round, 
refractive  bodies,  which  finally  escape  from  the  cyst, 
each  body  possessing  a  single,  long  flagellum,  of  very 
delicate  structure.  These  little  flagellates  are 
actively  motile,  increase  considerably  in  size,  and 
undergo  longitudinal  division  for  several  generations. 
At  the  end  of  this  period  of  reproduction  the  para- 


220  PARASITIC  AMOEBAE  OF  MAN. 

sites  become  motionless;  the  flagellum  disappears; 
the  border  of  the  spherical  body  remaining  begins  to 
undulate;  and  eventually  a  blunt,  well  defined  pseu- 
dopodium  appears,  and  the  parasite  enters  on  its 
amoebic  stage  of  existence. 

The  morphology  of  Paramceba  hominis  varies 
greatly  in  its  different  stages  of  development  and 
for  this  reason  it  will  be  necessary  to  describe  the 
morphology  of  each  stage,  i.e.,  the  amoebic  stage,  the 
encysted  stage,  and  the  flagellate  stage. 

The  Amoebic  Stage.— In  this  stage  the  parasites 
measure  from  10  to  25  microns  in  diameter,  the  aver- 
age measurement  being  from  18  to  20  microns. 
Amoeboid  motion  is  first  apparent  as  an  undulatory 
movement  of  the  periphery  of  the  parasite  followed 
by  the  projection  of  small,  bluntly  conical  pseudo- 
podia,  in  those  organisms  which  have  originated  from 
the  flagellate  forms,  but  in  those  amoebae  which  re- 
sult from  simple  division,  amoeboid  motion  is  first 
manifested  by  the  projection  of  pseudopodia.  In  the 
younger  organisms  amoeboid  motility  is  very  slug- 
gish, progressive  motion  being  absent,  as  a  rule, 
although  the  pseudopodia  may  be  projected  and  with- 
drawn with  considerable  rapidity;  in  the  larger  and 
older  amoebae  progressive  motion  is  quite  marked,  the 
ectoplasmic  pseudopodia  being  projected  rapidly  and 
the  endoplasm  flowing  into  them  immediately. 


FIG.  XXVIII. —  Diagram  of  the  life-cycle  of  Paramceba  hominis;  A, 
the  entire  life-cycle,  showing  the  amoeba  and  the  flagellate  stages,  as  well  as 
the  stage  of  encystment;  B,  reproduction  of  the  amcebic  stage  by  simple 
division  or  cycle  of  reproduction  by  simple  division;  C,  reproduction  of  the 
flagellate  stage  by  longitudinal  division  or  cycle  of  reproduction  by  longi- 
tudinal division. 


AMOEBAE  OF  THE  INTESTINAL  TRACT. 

When  motionless  no  distinction  can  be  made  be- 
tween the  ecto-  and  endoplasm,  but  when  moving, 
even  in  the  smallest  amoebse,  these  two  divisions  of 
the  cytoplasm  can  be  easily  distinguished,  the  endo- 
plasm being  more  refractive  than  the  ectoplasm  and 
apparently  of  greater  consistence.  The  endoplasm 
comprises  about  three-fourths  of  the  substance  of 
the  parasite  and  is  finely  granular  in  structure,  while 
the  ectoplasm  appears  homogeneous  in  structure  and 
of  very  slight  consistence.  The  endoplasm  may  con- 
tain bacteria,  diatoms,  crystals,  and  occasionally  one 
or  more  red  blood  corpuscles. 

The  greater  degree  of  refraction  of  the  endo- 
plasm of  Paramceba  hominis,  as  compared  with  the 
ectoplasm,  serves  to  distinguish  the  amoebic  stage  of 
this  parasite  from  Entamceba  histolytica  and  Enta- 
mceba  tetragena,  in  which  the  ectoplasm  is  more  re- 
fractive than  the  endoplasm;  and  from  Entamceba 
coli,  in  which  there  is  practically  no  distinction  be- 
tween the  ecto-  and  endoplasm. 

The  nucleus  can  be  easily  distinguished  in  even 
the  smallest  amoebse ;  it  is  a  refractive,  spherical  body, 
surrounded  by  a  rather  thick,  very  refractive,  granu- 
lar nuclear  membrane,  which  in  the  larger  organisms 
appears  to  be  composed  of  brightly  refractive  rods 
arranged  end-to-end  around  the  periphery  of  the  less 
refractive  nuclear  substance,  or  of  large  granules 


PARASITIC  AMOEBJE  OF  MAN. 

arranged  side  by  side.  There  is  no  visible  karyosome, 
but  a  few  very  minute  granules  of  chromatin  may 
sometimes  be  observed  in  the  hyaloplasm.  In  the  fully 
developed  amoebse  an  oval  body  may  be  observed  which 
lies  in  contact  with,  or  very  near,  the  nucleus,  and 
which  is  about  one-third  the  size  of  the  latter.  This 
body  undoubtedly  corresponds  to  the  cytoplasmic 
body  (Nebenkorper,  centrosome,  or  blepharoplast ) 
described  by  Schaudinn  in  Paramceba  eilhardi. 

A  nutritive  vacuole  is  not  present  in  this  species, 
so  far  as  I  have  been  able  to  determine,  although 
small  oval  bodies  are  sometimes  present  which  sug- 
gest vacuoles. 

In  reproduction  by  simple  division  the  cytoplasmic 
body  appears  to  divide  first,  followed  quickly  by  the 
division  of  the  nucleus,  and  finally  by  the  cytoplasm, 
two  daughter  amoeba?  being  thus  produced. 

In  the  amoebic  stage  the  parasite  stains  poorly, 
although  it  may  be  stained  with  Wright's  method, 
Heidenhain's  iron  hsematoxylin,  carbol-fuchsin,  Bor- 
rel  blue,  or  methylene  blue.  These  stains  do  not  dif- 
ferentiate the  ecto-  and  endoplasm,  but  the  nucleus 
stains  fairly  well  and,  when  the  organism  is  dividing, 
shows  well  marked  mitotic  figures.  With  Wright's 
stain  the  nucleus  appears  to  be  composed  almost  en- 
tirely of  chromatin,  staining  a  pink  or  reddish  violet, 
while  the  cytoplasmic  body  may  sometimes  be  dis- 


AMGEBJE  OF  THE  INTESTINAL  TRACT. 

tinguished  as  a  deep  violet  or  almost  black  mass, 
lying  in  contact  with  the  nucleus.  In  stained  speci- 
mens the  cytoplasmic  body  is  always  very  small  as 
compared  with  the  nucleus. 

The  Encysted  Stage. — For  the  observation  of 
the  process  of  encystment  it  is  necessary  to  examine 
fresh  preparations  as  the  cysts  do  not  stain  well. 

The  organisms  which  are  about  to  encyst  are 
generally  smaller  than  the  average  amoebae,  measur- 
ing from  15  to  18  microns  in  diameter,  and  appear 
more  granular  in  structure.  Amreboid  motility  is 
absent  and  the  ecto-  and  endoplasm  are  indistinguish- 
able. If  one  of  these  organisms  be  watched  it  will 
be  observed  that  it  suddenly  begins  to  rotate  quite 
rapidly  and  that  this  rotation  may  last  for  an  hour 
or  more,  although  it  generally  ceases  within  fifteen 
minutes.  The  rotation  is  in  one  direction  and  during 
this  process  the  cyst  wall  is  formed,  for  when  it  has 
ceased  it  will  be  observed  that  the  organism  is  sur- 
rounded by  a  double-outlined,  refractive  capsule, 
which  sometimes  appears  slightly  mammillated.  Dur- 
ing rotation  the  organism  contracts  somewhat,  most 
of  the  cysts  measuring  about  15  microns  in  diameter. 

In  the  early  stage  of  encystment  the  nucleus  can 
be  distinguished,  situated  to  one  side  of  the  centre 
of  the  organism,  and  the  cytoplasmic  body  may  also 
be  visible,  lying  in  contact  with  the  nucleus.  Both 


PARASITIC  AMOEBAE  OF  MAN. 

soon  disappear,  however,  and  the  cyst  becomes  filled 
with  refractive  granules  due  to  the  division  of  the 
cytoplasmic  body  and  the  nucleus.  At  a  later  stage 
of  development  the  cysts  appear  to  be  crowded  with 
small  spherical  bodies,  which  are  refractive  and  some- 
times appear  to  move  about  within  the  cyst  wall,  but 
this  motion  may  be  molecular  in  nature.  At  the  very 
latest  stage  of  development  within  the  cyst,  the  out- 
line of  the  young  flagellates  may  be  distinguished, 
most  of  them  appearing  spherical  in  shape  and  a 
dull  gray  in  color,  but  the  flagellum  cannot  be  dis- 
tinguished while  they  are  still  contained  within  the 
cyst. 

The  Flagellate  Stage. — I  have  not  been  able  to 
observe  the  escape  of  the  young  flagellates  from  the 
mother  cyst,  but  groups  of  these  organisms  are  fre- 
quently observed,  surrounded  by  the  ruptured  cyst 
wall  and  arranged  in  spherical  masses  corresponding 
in  size  with  the  original  cyst.  It  is  very  evident  that 
these  organisms  have  developed  within  the  cyst  and 
they  very  soon  assume  the  typical  appearance 
presented  by  the  flagellate  stage  of  the  parasite. 

In  the  youngest  stage  of  development,  i.e.,  just 
after  liberation  from  the  cyst,  the  swarm-spores  do 
not  appear  to  possess  a  flagellum;  they  are  very 
small,  measuring  from  3  to  6  microns  in  diameter, 
are  spherical  in  shape,  and  have  a  finely  granular 


AMOEBAE  OF  THE  INTESTINAL  TRACT.     225 

cytoplasm  in  which  the  nucleus  is  not  well  differen- 
tiated. If  one  watches  these  young  amoebae  it  will 
be  observed  that  they  soon  become  motile,  a  very 
delicate  flagellum  appearing  at  some  portion  of  the 
periphery;  they  disengage  themselves  from  the  ma- 
terial in  which  they  often  appear  to  be  imbedded, 
and  move  about  in  a  rapid,  jerky  manner,  propelled 
by  the  flagellum,  although  at  times  the  latter  may 
appear  to  draw  the  organism  forward.  The  very 
young  forms  do  not  stain  well  with  any  method 
which  I  have  tried. 

The  flagellate  forms  grow  rapidly  and  when  fully 
developed  measure  from  10  to  20  microns  in  diameter. 
They  are  spherical  in  shape  except  at  that  portion  of 
the  periphery  where  the  flagellum  is  attached,  where 
the  cytoplasm  is  continued  into  the  flagellum,  thus 
giving  the  organism  a  pear-shaped  appearance.  The 
flagellum  is  from  three  to  four  times  as  long  as  the 
diameter  of  the  parasite,  and  tapers  very  rapidly, 
the  outer  three-fourths  being  so  extremely  delicate  as 
to  require  the  most  careful  focussing  to  demonstrate 
it.  The  nucleus  in  most  instances  is  situated  near 
the  origin  of  the  flagellum,  which  generally  appears 
to  be  situated  posteriorly. 

The  cytoplasm  of  the  parasite  at  this  stage  of 
development  is  finely  granular  in  structure  and  con- 
tains a  small,  well  defined  nucleus,  and  a  minute 


15 


226  PARASITIC  AMOEBJS  OF  MAN. 

cytoplasmic  body,  the  Nebenkorper  of  Schaudinn. 
The  nucleus  is  spherical  in  shape,  and  has  a  delicate 
refractile  nuclear  membrane.  The  cytoplasmic  body 
lies  in  contact  with  the  nucleus,  measures  about  2 
microns  in  diameter,  and  is  somewhat  refractile.  In 
rare  instances  the  cytoplasm  may  contain  one  or  two 
red  blood  corpuscles,  so  that  it  is  evident  that  phago- 
cytosis of  these  cells  is  not  confined  to  the  amoebic 
stage  of  development. 

Reproduction  by  longitudinal  division  may  some- 
times be  observed.  When  this  is  occurring  the 
organisms  present  two  nuclei  and  at  certain  stages 
one  can  observe  the  partial  division  of  the  flagellum 
into  two  portions,  the  splitting  of  the  flagellum  being 
first  noticeable  at  its  point  of  attachment  to  the  body 
of  the  parasite.  That  these  forms  are  not  conjugat- 
ing organisms  is  proved  by  the  fact  that  the  division 
of  the  nucleus  may  be  observed  before  there  is  any 
division  of  the  flagellum,  the  parasite  at  this  stage 
showing  two  nuclei  and  but  one  flagellum.  It  is 
probable  that  the  division  of  the  nucleus  is  preceded 
by  the  division  of  the  cytoplasmic  body,  but  I  have 
not  been  able  to  satisfy  myself  that  this  is  so.  After 
the  division  of  the  nucleus  is  complete,  the  cytoplasm, 
as  well  as  the  flagellum,  divides  longitudinally,  thus 
forming  two  parasites. 

After  reproduction  in  this  manner  has  occurred 


FIG.  XXIX. — Various  forms  of  the  amoebic,  cystic,  and  flagellate  stages 
of  Paramceba  hominis.  1  to  13,  various  forms  of  the  amoebic  stage  of 
Paramosba  hominis.  Note  the  well-defined  nucleus,  the  oval  or  round 
cytoplasmic  body  or  Nebenkbrper  in  contact  with  the  nucleus  in  some  of  the 
forms;  the  granular  protoplasm;  the  differentiation  of  the  ectoplasm  and 
endoplasm;  the  blunt  pseudopodia.  At  12  is  shown  a  form  containing  a 
red  blood-corpuscle;  14,  15,  and  16  are  examples  of  the  encysted  stage, 
showing  the  division  of  the  nucleus  and  cytoplasmic  body  into  the  swarm 
spores;  17,  young  parasites  just  after  leaving  the  cyst.  Some  show  a  short 
flagellum,  while  others  do  not  show  the  flagellum;  18  and  19  are  developing 
flagellate  forms  of  Paramceba  hominis,  showing  the  flagellum,  nucleus,  and 
cytoplasmic  body;  20,  fully  developed  flagellate  forms,  one  of  which  is 
undergoing  longitudinal  division;  21,  flagellate  forms  just  after  the  disap- 
pearance of  the  flagellum,  one  of  which  already  shows  the  typical  amosbic 
stage. 


AMOEBAE  OF  THE  INTESTINAL  TRACT. 

for  several  generations  the  parasites  become  spherical 
in  shape,  the  flagellum  is  lost,  and  the  organisms  enter 
upon  the  amoebic  stage  of  existence. 

Even  when  well  developed  the  flagellate  forms 
stain  very  poorly,  but  with  Wright's  stain  I  have 
been  able  at  times  to  get  a  clear  differentiation  be- 
tween the  cytoplasm  and  the  nucleus,  the  cytoplasm 
staining  a  deep  blue,  while  the  nucleus  stains  a  reddish 
violet.  With  this  stain  the  cytoplasmic  body  ap- 
pears almost  black  in  color  and  is  not  well  differen- 
tiated. The  flagellum  may  sometimes  be  stained  a 
very  dim  pink,  if  the  stain  be  allowed  to  act  for  an 
hour  or  more. 

RELATION  TO  DISEASE. — I  have  not  been  able  to 
produce  an  infection  in  animals  with  this  parasite,  but 
from  the  clinical  symptoms  present  in  the  patients 
infected  with  Paramceba  hominis  and  the  fact  that 
recovery  quickly  follows  the  disappearance  of  the  par- 
asite in  all  the  cases  I  have  observed,  I  believe  that 
it  is  justifiable  to  conclude  that  it  may  cause  a  form 
of  chronic  diarrhoea,  characterized  by  acute  exacerba- 
tions alternating  with  periods  of  constipation.  All  of 
the  patients  were  suffering  from  diarrhoea  at  the  time 
the  parasites  were  found  in  their  feces,  although  in 
one  instance  the  condition  was  complicated  by  a  severe 
dysentery  due  to  Entamoeba  Mstolytica.  Omitting 
this  case,  we  had  8  patients  in  whom  the  presence  of 


PARASITIC  AMOEBAE  OF  MAN. 

Paramoeba  hominis  was  accompanied  by  symptoms  of 
a  severe  diarrhoea,  alternating  with  periods  of  con- 
stipation. In  five  of  the  patients  the  f eces  contained 
a  small  amount  of  blood  and  mucus,  while  in  two 
Trichomonas  hominis  was  present  in  small  numbers. 
It  will  thus  be  seen  that  in  eight  of  the  cases  Para- 
mceba hominis  was  the  only  protozoon  present  which 
could  be  looked  on  as  of  possible  etiological  signifi- 
cance, the  two  patients  showing  trichomonads  having 
them  in  too  small  numbers  to  suggest  any  relation 
between  them  and  the  symptoms  present.  Treatment 
by  irrigation  of  the  bowel  resulted  in  the  disappear- 
ance of  the  parasites,  and,  with  them,  of  the  diarrhoea, 
and  none  of  the  patients  have  relapsed,  so  far  as  I 
have  been  able  to  determine.  Thus  the  clinical  evi- 
dence points  to  Paramceba  hominis  as  the  cause  of 
the  diarrhoea,  and  I  believe  that  we  may  safely  regard 
this  parasite  as  belonging  to  the  pathogenic  protozoa. 
Differential  Diagnosis. — In  the  amoebic  stage  of 
development  Paramceba  hominis  might  be  confused 
with  Entamceba  coli,  Entamceba  histolytica,  or  Enta- 
mceba  tetragena.  If  it  be  remembered  that  the  endo- 
plasm  of  this  species  is  more  refractive  than  is  the 
•ectoplasm,  it  will  be  easy  to  differentiate  it  from 
other  intestinal  amoebae.  However,  the  occurrence  of 
the  peculiar  cysts  and  of  the  flagellate  stage  of  devel- 
opment, at  the  same  time,  should  enable  one  to  diag- 


AMCEB.E  OF  THE  INTESTINAL  TRACT. 

nose  this  species  of  amoeba  with  but  little  difficulty^ 
When  one  observes  in  feces  the  peculiar  rotating 
organisms  which  I  have  described,  one  may  be  sure 
that  Paramceba  hominis  is  present. 

In  the  flagellate  stage  of  development  the  only 
organism  occurring  in  the  feces  which  might  be  con- 
fused with  Paramceba  hominis  is  Trichomonas  hom- 
inis, because  of  certain  peculiarities  in  the  develop- 
ment of  the  latter.  Trichomonas  hominis  is  fre- 
quently observed  in  the  resting  stage,  when  it  is 
spherical  in  shape,  and  appears  to  possess  a  limited 
degree  of  amoeboid  motion.  However,  it  is  much 
smaller  than  Paramceba  hominis  and  never  shows  the 
active  progressive  motion  observed  in  the  latter.  The 
flagellate  stage  of  Paramceba  hominis  is  distinguished 
from  active  trichomonads  by  the  absence  of  an  un- 
dulating membrane,  the  presence  of  but  one  flagellum, 
and  the  more  spherical  form  of  the  organism. 

The  simultaneous  occurrence  in  the  feces  of  the 
amoebic  stage,  the  encysted  stage,  and  the  flagellate 
stage  of  Paramceba  hominis,  is  characteristic  of  this 
organism,  and  no  difficulty  should  be  experienced 
by  one  who  is  well  acquainted  with  intestinal  protozoa,, 
in  differentiating  this  species  from  other  parasites 
which  infest  the  intestine  of  man. 


VII. 

THE  AMOEBA  OF  THE  MOUTH. 

SEVERAL  observers  have  noted  the  occurrence  of 
amoebae  in  the  mouth,  the  parasites  being  found  in 
the  tartar  around  the  roots  of  the  teeth,  or  in  ma- 
terial from  the  cavities  of  carious  teeth.  As  long 
ago  as  1862,  Steinberg  described  an  amoeba  in  the 
tartar  removed  from  teeth,  to  which  the  name  Amoeba 
buccalis  has  been  given,  and  it  is  more  than  probable 
that  this  species  is  identical  with  the  one  described 
very  fully  by  Prowazek,  who  placed  it  in  the  genus 
Entamceba. 

In  1904  Prowazek  investigated  the  amoeba?  occur- 
ring in  the  human  mouth  and  decided  that  they  be- 
long to  a  distinct  species.  He  gave  a  detailed  de- 
scription of  this  parasite  and  named  it  Entamceba 
buccalis.  He  found  it  present  in  carious  teeth,  and 
considers  that  all  amoebae  which  have  been  described 
as  occurring  in  the  mouth  belong  to  this  species. 

The  GEOGRAPHICAL  DISTRIBUTION  of  this  species 
is  world-wide.  I  have  observed  it  in  the  mouth  of 
patients  in  the  United  States  and  the  Philippines, 
and  it  is  probable  that  a  careful  examination  will 
demonstrate  that  it  occurs  in  almost  every  locality. 

230 


AMCEBJS  OF  THE  MOUTH.  231 

MORPHOLOGY. — This  species  is  relatively  small, 
measuring  from  6  to  32  microns  in  diameter,  the 
average  measurement  being  15  microns.  It  has  a 
distinct  ectoplasm,  while  the  endoplasm  appears  quite 
granular  and  is  filled  with  vacuoles.  The  ectoplasm 
is  seldom  visible  unless  the  organism  is  moving,  when 
it  appears  clear  and  hyaline  and  is  little  more  re- 
fractive than  the  endoplasm.  The  nucleus  is  well 
defined,  spherical  or  oval  in  shape,  and  surrounded 
by  a  thick  greenish  nuclear  membrane  containing 
much  refractive  material  resembling  chromatin.  A 
small  centriole,  surrounded  by  a  clear  area  and  a 
small  chromatin  zone,  is  situated  near  the  centre  of 
the  nucleus. 

MotiUty  is  sluggish  in  character  and  is  produced 
by  the  extrusion  of  short,  blunt,  ectoplasmic  pseudo- 
podia  into  which  the  endoplasm  flows.  The  motility 
is  not  as  marked  as  in  Entamceba  histolytica  or 
Entamceba  tetragena,  but  is  more  marked  than  in 
Entamceba  coli. 

Reproduction  occurs  by  simple  division,  the 
nucleus  dividing  by  mitosis,  and  a  nuclear  spindle  is 
often  observed.  Schizogony  has  been  described  as 
occurring  in  this  species,  the  nucleus  distributing 
chromatin  to  the  cytoplasm,  which  eventually  divides 
into  small  amoebae,  each  containing  a  small  amount 
of  nuclear  chromatin. 


232  PARASITIC  AMOEBAE  OF  MAN. 

This  organism  has  not  been  cultivated  artificially 
so  far  as  I  am  aware. 

There  is  no  experimental  evidence  connecting 
Entamceba  buccalis  with  disease.  It  is  very  doubtful 
if  it  has  anything  to  do  with  caries  of  the  teeth, 
although  it  is  most  frequently  encountered  in  the 
cavities  of  carious  teeth.  However,  it  may  be  fre- 
quently demonstrated  in  material  scraped  from  the 
roots  of  perfectly  normal  teeth,  so  that,  as  far  as  the 
evidence  goes,  we  must  regard  Entamceba  buccalis  as 
only  a  secondary  invader  of  the  tissues. 

The  amoebae  known  as  Entamceba  gingivalis,  de- 
scribed by  Gros,  in  1849,  and  Entamceba  dentalis, 
described  by  Grassi  in  1879,  are  in  all  probability 
identical  with  Entamceba  buccalis  and  do  not  merit 
a  separate  description. 


^ 

o 


FIG.  XXX. — Entamceba  buccalis.  A,  living  form  showing  the  char- 
acter of  the  ecto-  and  endoplasm  and  the  nucleus;  B,  form  stained  with  iron 
hsematoxylin,  showing  the  reticulative  appearance  of  the  nucleus  and  endo- 
plasm; C,  Entamceba  buccalis  stained  with  the  Giemsa  stain,  showing  the 
reticulative  structure  of  the  nucleus  and  endoplasm.  (After  Hartmann.) 


VIII. 

THE  AMOEBA  OF  THE  GENITO-URINARY  TRACT. 

IN  1883  Baelz  described  an  amoeba  occurring  in 
bloody  urine  which  measured  from  23  to  50  microns 
in  diameter  and  was  actively  motile,  the  pseudopodia 
being  short  and  blunt.  The  cytoplasm  of  this 
organism  was  granular  in  appearance  and  the  para- 
site was  phagocytic  for  red  blood  corpuscles.  En- 
cysted forms  were  also  observed. 

To  this  organism  the  name  Entamceba  urogenitalis 
has  been  given  and  Jiirgens,  Posner,  and  Kartulis 
have  described  similar  amoebae  occurring  in  the  urine. 
It  is  still  undecided  whether  this  organism  is  entitled 
to  specific  rank,  most  authorities  believing  that  the 
amoebae  described  by  these  authors  were  either  Enta- 
mceba histolytica  or  Entamceba  tetragena.  There  is 
no  reason  why  any  of  the  species  occurring  in  the 
intestine  should  not  occasionally  be  found  in  the  urine, 
reaching  it  through  fistulas  between  the  bladder  and 
intestine,  which  may  occur  in  cases  of  amoebic  dysen- 
tery, or  intestinal  amoebae  might  reach  the  kidney  or 
bladder  through  the  surrounding  tissues  or  through 
the  blood  stream.  I  have  observed  one  instance  of 
infection  of  the  bladder  with  Entamceba  histolytica, 
autopsy  showing  a  minute  fistula  between  the 
ulcerated  intestine  and  the  bladder. 

233 


IX. 

AMOEBAE  OCCURRING  IN  EXUDATIONS,  ABSCESSES, 
AND  IN  THE  LUNGS. 

IJIMA  described  amoebae  occurring  in  serous 
fluid  obtained  from  a  woman  suffering  from  peri- 
tonitis due  to  an  endothelioma,  to  which  he  gave  the 
name  of  Entamceba  miurai.  He  described  this 
organism  as  measuring  from  15  to  18  microns  in 
diameter,  and  spherical  or  oval  in  shape,  with  a 
pseudopodium  at  one  end  covered  with  cilia.  The 
cytoplasm  is  described  as  finely  granular  and  no  dis- 
tinction was  noted  between  the  ectoplasm  and  endo- 
plasm.  From  1  to  3  nuclei  were  observed,  but  no 
data  are  given  regarding  the  methods  of  reproduction. 

It  is  generally  believed  that  Ijima  mistook  cells 
present  in  the  serous  exudation  for  amoeba?  and  his 
observations  have  not  been  confirmed.  I  do  not  be- 
lieve that  Entamceba  miurai  can  be  considered  a  valid 
species. 

In  1892  Flexner  described  an  amoeba  occurring 
in  the  pus  of  an  abscess  in  the  oral  cavity,  and  in 
1893  a  similar  organism  was  described  by  Kartulis 
in  abscesses  of  the  lower  jaw,  and  to  this  parasite 
the  name  Entamoeba  kartulm  has  been  given.  The 
description  given  by  Flexner  and  Kartulis  could  be 

234 


AMCEB.E  OCCURRING  IN  EXUDATIONS,  ETC.       235 

in  part  applied  to  either  Entamoeba  Mstolytica  or 
Entamceba  buccalis,  and  it  is  doubtful  if  this  species 
can  be  considered  valid.  It  is  well  known  that  under 
certain  conditions  Entamoeba  Mstolytica  enters  the 
blood  stream  and  may  be  carried  to  distant  tissues 
where  it  may  produce  abscess  formation  and  abscesses 
due  to  this  organism  have  been  found  in  the  brain, 
lungs  and  kidneys.  From  the  description  given  of 
Entamoeba  kartulisi  it  is  very  probable  that  it  is 
identical  with  Entamoeba  Mstolytica,  and  that  the  ab- 
scesses in  which  it  was  found  were  caused  by  the 
latter  organism. 

Artault,  in  1898,  described  an  amoeba  occurring  in 
the  lungs  which  is  now  known  as  Entamoeba  pul- 
monalis.  The  description  of  this  species  is  quite 
similar  to  that  of  Entamoeba  Mstolytica,  and  in  all 
probability  the  latter  organism  was  the  one  observed 
by  Artault.  Entamoeba  pulmonalis  cannot  be  con- 
sidered a  valid  species. 


REFERENCES 


ARTAULT,  ST.     Flore  et  faune  d.  cav.  pulmon,  Amoeba  pulmonalis, 

sp.  n.     Arch,  de  parasit.,  1898,  I,  p.  275. 
ASHBURN,  P.  M.,  and  CRAIG,  C.  F.     Entamceba  coli,  etc.     The 

Military  Surgeon,  1907,  I,  p.  21. 

BAELZ,  E.  Ueber  einige  neue  Parasiten  des   Menschen.     Berl. 

klin.  Wochenschr.,  1883,  10,  p.  287. 
BENSEN,    W.    Die    Darmprotozoon    des    Menschen.      Arch.    f. 

Schiffs-  u.  Tropen-Hyg.,  1908,  12,  p.  661. 
BERNDT.     Deutsche  Ztschr.  f.  Chirurgie,  1894,  40,  p.  163. 
BILLET,  A.     Sur  un  cas  de  dysenteric  "  nostras  "  a  ainibes.     C. 

R.  Soc.  Biol.,  1907,  Ixii,  p.  1232. 
BLANCHARD,  R.     Traite  de  Zool.  medic.     T.  I.     Paris,  1885,  p. 

33.     Idem.     Maladies  parasit.      Paris,  1895,  p.  658. 
BRAUN,    M.      Tierische    Parasiten    des    Menschen.      Wiirzburg, 

1908,  p.  33. 
BRAUN,  M.,  and  LUHE,  A'.     Handbook  of  Practical  Parasitology, 

New  York,  1910. 
BROWN,  W.  C.     Amoebic  or  Tropical  Dysentery,  London,  1910. 

CAHEN.     Deutsche  med.   Wochenschr.,   1891,    17,  p.   843. 
CALANDRUCCIO.     Anim.  par.  dell'  nomo  in  Sicilia.     Atti.  Accad. 

Giorn.,  1890,  2,  p.  95. 

CALKINS,  G.     Protozoology.     Philadelphia,  1909. 
CASAGRANDI,    O.,    and    BARBAGALLO,    P.     Recersche    Biolog.,    e 

cliniche  sell'  amoeba  coli   (Loesch).     Boll.  Accad.  Groinea. 

sc.  nat.    Catania,  1895,  xli,  pp.  7-17. 

2S7 


238  REFERENCES 

Idem.  Sui  terreni  di  coltura  delle  amobe.  Ref.  med.,  1896, 
157,  p.  7477. 

Idem.  Entamoebae  hominis  a  amoeba  coli  (Loesch).  Amali 
d'Igiene  sperimentale,  1897,  V.  I,  p.  103. 

Idem.  Ueber  die  Kultur  von.  Amoeben.  Centralbl.  f.  Bakt., 
etc.,  1897,  1  Abts.  xxi,  p.  579. 

CASTELLANI,  A.  Observations  on  some  Protozoa  found  in  Hu- 
man Faeces.  Centralbl.  f.  Bakt.,  etc.,  1905,  1,  xxxviii,  p.  66. 

CELLI,  A.,  and  FIOCCA,  R.  Beitrage  zur  Amoebenforschung. 
Centralbl.  f.  Bakt.,  etc.,  1894,  1,  xv,  p.  470. 

Idem.  Beitrage  zur  Amcebenforschung  u.  d.  Klassification  der 
Amceben,  etc.  Ibid.,  1894,  1,  xvi,  p.  329. 

Idem.  Richerche  interno  alia  biologia  delle  amebe.  Bull.  d.  r. 
Accad.  med.  di  Roma,  1895,  xxii,  p.  285. 

Idem.  Ueber  die  Aetiologie  der  Dysenteric.  Centralbl.  f.  Bakt., 
etc.,  1895,  1,  xvii,  p.  309. 

COUNCILMAN  and  LAFLEUR.  Amoebic  Dysentery.  Johns  Hop- 
kins Hospital  Reports,  ii,  pp.  395-548,  1891. 

CRAIG,  CHAS.  F.  Observations  on  Amoeba  coli,  etc.  Med.  News, 
1901,  Ixxviii,  11,  p.  414. 

Idem.  Classification  of  Amoeba  coli.  Am.  Med.,  Phila.,  1904, 
viii,  p.  185. 

Idem.  The  Complications  of  Amoebic  and  Specific  Dysentery 
as  Observed  at  Autopsy;  an  Analysis  of  One  Hundred  and 
Twenty  Cases.  Am.  Jour.  Med.  Sci.,  n.  ser.,  1904,  cxxvii, 
p.  145. 

Idem.  The  Life  Cycle  of  Amoeba  coli  in  the  Human  Body. 
Am.  Med.,  Phila.,  1904,  vii,  p.  299. 

Idem.  The  Pathology  of  Chronic  Specific  Dysentery  of  Tropical 
Origin.  Jour.  Assoc.  Mil.  Surg.,  1904,  xiv,  p.  353. 

Idem.  Observations  upon  Amoebae  Infecting  the  Human  In- 
testine, with  a  Description  of  two  Species,  Entamosba  coli 


REFERENCES  239 

and  Entamoeba  Dysenteriae.     Am.  Med.,  Phila.,  1905,  ix,  pp. 

854;    897;    937. 
Idem.     The  Etiology  and  Pathology  of  Amoebic  Infection  of  the 

Intestine  and  Liver.     Internat.  Clin.,  Phila.,  1905,  14  Ser., 

V.  4,  pp.  242-298. 
Idem.     A  New  Intestinal  Parasite  of  Man;    Paramoeba  hominis. 

Am.  Jour.  Med.  Sci.,  V.  Ser.,  1906,  cxxxii,  8,  p.  214. 
Idem.     Studies  upon  the  Amoebae  in  the  Intestine  of  Man.  Jour. 

Infec.  Diseases,  1908,  5,  3,  pp.  324-377. 
Idem.     Further  Observations  on  Paramceba  hominis,  an  Intestinal 

Parasite  of  Man.     Arch.  Internal  Med.,  1910,  6,  1,  pp.  1-11. 
Idem.     Entamoeba   tetragena   as   a   Cause   of   Dysentery   in   the 

Philippine    Islands.      Arch.    Internal    Med.,    191 1^    vii,    3, 

p.  362. 

CUNNINGHAM,  D.  D.     On  the  Development  of  Certain  Micro- 
scopic Organisms  Occurring  in  the  Intestinal  Canal.     Quar. 

Jour.  Micro.  Sci.,  1881,  V.  Ser.  21,  Ixxii,  p.  234. 

DOCK,    G.     Observations    on    Amoeba    coli    in    Dysentery    and 

Abscess    of    the    Liver.      Texas    Med.    Jour.,    1890-91,    vi, 

p.   419. 
DOFLEIN,  F.     Lehrbuch  der  Protozoenkunde.     Berlin,  1909*  ?• 

500. 
DOPTER,  C.     Sur  Quelques  Points  relatifs  a  Faction  pathogene 

de  1'amibe  dysenterique.     Ann.   Instit.   Pasteur,   1905,  xix, 

p.  417. 

Idem.     Les  dysenteries.     Paris,  1908. 
DUTROLEAU.     Maladies   en   Pays  chauds.     Paris,   1868. 

ELMASSIAN.     Entamoeba  minuta,  etc.     Centralbl.   f.   Bakt.,  etc., 
1909,  1,  Abt.  Orig.  52,  p.  335. 


240  REFERENCES 

FANTHAM,  H.  B.  On  the  Amoebae  Parasitic  in  the  Human  In- 
testine, etc.  Ann.  Trop.  Med.  and  Parasitology,  Ser.  T.  M. 
1911,  V.  1,  p.  111. 

FLEXNER,  S.  Dysentery.  System  of  Medicine,  Allbutt  and 
Rolleston,  Vol.  II,  Part  2,  1907,  London,  p.  477. 

FROSCH,  P.  Zur  Frage  der  Reinzuechtung  der  amceben.  Cen- 
tralbl.  f.  Bakt.,  etc.,  1897,  xxi,  24-25,  pp.  926-932. 

FUTCHER,  T.  B.  A  Study  of  the  Cases  of  Amoebic  Dysentery 
Occurring  at  the  Johns  Hopkins  Hospital.  Jour.  Am.  Med. 
Assoc.,  1903,  xii,  p.  480. 

GASSER,  J.     Note  sur  les  causes  de  la  dysenteric.     Arch.  Med. 

Exp.,  etc.,  1895,  ii,  p.  198. 
GAUDUCHEAU,    A.     An    Experimental    Production    of    Amoebic 

Dysentery,  etc.     Jour.  Trop.  Med.,  1906,  ix,  p.  15. 
Idem.     Note    preliminaire    sur    la    culture    et    la    function    bac- 

teriolytique    d'un    protozaire    amiboide.      Gazette    Hebdom- 

adaire   d.    sci.    med.,    1907,    20,    17,   p.    193.      Also    Bull. 

parasit.    Exotique,    1909,   ii,   p.    247. 
Idem.     Culture    d'une    amibe    dysenterique    (e.    phagocytoides) . 

C.  R.  Soc.  Biol.,  1908,  Ixiv,  p.  493. 
GRASSI,   B.     Die    Protoi   parasite   e   specialmente   di   quelli   che 

sono  nell'  uomo.     Gaz.  Med.  Ital.,  Lombardia,  1879,  p.  445. 
Idem.     Contributione   allo   Studio   delle   amibe.     Rendic.   d.    R. 

inst.  Lombardia,  1881,  2,  xiv,  p.  353. 
GROS,  A.  Beobacht.  uber  Amcebenenteritis.     Arch.  f.  klin.  Med., 

1903,  Ixxvi,  p.  429. 

HARRIS,  H.  F.  Some  observations  on  a  Method  of  Multiplica- 
tion of  the  Amoeba  dysenteriae  (Amoeba  coli).  Med.  News, 
1894,  Ixv,  21,  p.  567. 


REFERENCES  241 

Idem.     On  the  Alterations  Produced  in  the  Large  Intestine  of 

Dogs    by    the    Amoeba    coli,    etc.     Hatfield    Priae    Essay. 

Phila.,  1901. 
Idem.     Experimentell  bei  Hunden  erzeugte  Dysenteria.     Arch. 

Path.  Anat.  Berl.,   1901,  clxvi,  p.   67. 

Idem.     Amoebic  Dysentery.     Am.  Jour.  Med.  Sci.,  April,  1908. 
HARTMANN,  M.     Eine  neue  Dysenteric  amobe.     Beiheft  z.  Arch. 

f.  Schiffs-  u.  Tropen  Hyg.,  1908,  v,  p.  117. 
Idem.     Untersuchungen     iiber    parasitischen     amoben     1.     Ent. 

histolytica     Sehandusic.     Arch.     f.     Protistenkunde,     1909, 

xviii,  p.  207. 
HARTMANN,  M.,  and  PROWAZEK,  S.     Blepharoplast,  Caryosome 

und  Centrosom.     Arch.  f.  Protistenk.,  1907,  x,  p.  312. 
HLAVA.— Ueber  die  Dysenteric.     Centralbl.  f.  Bakt,  etc.,  1887, 

1   Abt.  X,  p.  537. 

IJIMA,  J.  On  a  New  Rhizopod  Parasite  of  Man.  Annot.  Zool., 
Japan,  1898,  ii,  3,  p.  85.  Ref.  in  Centralbl.  f.  Bakt.,  etc., 
1899,  xxv,  p.  885. 

JAEGER,  H.  Untersuchungen  u.  Amoeben-dysenteric  in  Ost- 
preussen.  Deutsche  med.  Wochenschr.,  1902,  27,  p.  208. 

JURGENS.  Zur  Kenntniss  der  Darmamoeben  und  der  Amoeben- 
enteritis.  Veroff.  a.  d.  Geb.  d.  Milit-Sanitatswes.,  Berl., 
1902,  20,  p.  110. 

KARTULIS.  Ueber  Riesen  Amoeben  bei  chromischer  Darment- 
ziindung  der  Aegypten.  Virch.  Arch.  f.  path.  Anat.,  1885, 
xcix,  p.  145. 

Idem.  Zur  A'etiologie  der  Dysenteric  in  Aegypten.  Virch, 
Archiv  f.  path.  Anat.,  1886,  ex,  p.  521. 

16 


242  REFERENCES 

Idem.     Zur  Aetiologie  der   Leberabscesse.     Centralbl.   f.   Bakt., 

etc.,  1887,  1  Abt.  ii,  p.  745. 
Idem.      Ueber  welters      Verbreitungsgebiete      der      Dysenteric 

amoeben.     Ibid.,  1890,  1  Abt.  vii,  p.  54. 
Idem.     Einiges  ueber  die  Pathogenese  des  Dysenteric  amoeben. 

Ibid.,  1891,  1  Abt.  ix,  p.  365. 
Idem.     Die   amcebendysenterie.     Kolle  u.   Wassermann's   Hand- 

buch  d.  pathogenen  Micro-organismen,  1895,  1,  347. 
Idem.    Dysenteric.    Spec.    Path,    and    Flue.,    Nothnagel.     Ill, 

Wien,  1896. 
Idem.     Gehirnabscesse     nach     dysenterischen      Leberabscessen. 

Centralbl.  f.  Bakt.,  etc.,  1  Abt.  xxxvii,  4,  527. 
KOCH,   R.     Arbeit,   a.   d.   Gesondheitsamte.     No.   3,   1887. 
KOIDZUMI,    M.     On   a   New    Parasitic   Amoeba,    Entamoeba    nip- 

ponica.     Centralbl.  f.  Bakt.,  1909,  1,  Orig.  li,  p.  650. 
KOVACS,    F.    Beob.    u.    vers.    ut    d.    sog.    Amoeben-Dysenteric. 

Zeitschr.  f.  Heilkde.,  1892,  xiii,  p.  509. 
KRUSE,    W.,    and    PASQUALE,   A.     Studium    des    Dysenteric   und 

d.    Leberabscesse.      Deutsche   med.   Wochenschr.,    1893,    15, 

1,   354;     16,  p.   368. 
Idem.      Unters.   ueber    Dysenteric    u.    Leberabscesse.      Zeitschr. 

f.  Hyg.,  1894,  xvi,  pp.  1-149. 

LAMBL.     Beob.   u.   Studien   aus   dem   Franz-Joseph-Kinderspital 

in  Prag,  I860,  i,  p.   362. 
LESAGE,  A.     Culture  de  1'amibe  de  la  dysenteric  des  pays  chauds. 

Ann.  Inst.  Pasteur,  1905,  xviii,  pp.  9-1 6. 
Idem.     Culture  du  parasite  de  1'amibiase,  humaine.     C.  R.  Soc. 

Biol.,  1909,  Ixii,  p.   1157. 
Idem.     L'amibiase  chez   le  chat.     C.   R.   Soc.   Biol.,   1909,  Ixii, 

p.  1191. 
Idem.     Note    sur    1'Entamibe    d.    la    dysenteric    amibiene,    etc. 

Bull.  d.  FSoc.  d.   Path.  Exotique,   1908,   1,  p.   104. 


REFERENCES  243" 

LEWIS  and  CUNNINGHAM.     Ann.  Rep.  Sanit.  Commis.  Govt.  of 

India.     Calcutta,  1870. 
LOESCH,  F.     Massenk.   Entw.  v.  Amoeben  in  Dickdarm.   Virch^ 

Arch.  f.  path.  Anat.,  1875,  Ixv,  p.  196. 

MARCHOUX.     Note  sur  la  dysenteric.     C.   R.  Soc.  Biol.,  Paris, 

1899,  1,  p.  870. 
MARSHALL.     The  Amoeba  dysenteriae.     Brit.  M.  Jour.,  1899,  1, 

1386. 
MASSIUTIN.     Ueber  die  Amoeben   als   Parasiten  des  Dickdarms. 

Vratch,  1889,  x,  25;    Centralbl.  f.  Bakt.,  1889,  1  Abt.  vi, 

p.  451. 
MINCHIN.     The    Protozoa.     System   of    Medicine,   Allbutt    and 

Rolleston,  London,  1907,  Vol.  2,  Part  II. 
MIURA,    K.     Mitteilg.    d.    me    Fakult.    d.    kaiserl.    Japan    Univ. 

Tokio,  1900,  1,  pp.   1-18. 
MOUTON,  H.     Recherches  sur  la  digestion  chez  les  amibes,  etc. 

Ann.  Inst.  Pasteur,  1902,  xvi,  p.  457. 
MUSGRAVE,  W.  E.,  and  CLEGG,  M.  T.     Amebas ;   their  Cultivation 

and     Etiological     Significance.       Bureau     of     Govt.     Lab. 

Biological  Lab.,  1904,  18,  Manila,  P.  I. 
Idem.      The    Cultivation    and    Pathogenesis    of   Amoebae.      PhiL 

Jour.  Sci.,   1906,  1,  p.  909. 

NAKAGAWA.     Endemic  Dysentery  in  Formosa.     Mitteil.  de  med. 

GeselL,  Tokio,   1907. 
Noc,    F.     Sur   la   Dysenteric   Amibiene   en    Cochinchine.     Ann.. 

Inst.  Pasteur,  1909,  xxiii,  p.  177. 
NORMAND.     Note  sur  deux  cas  de  colite  parasite.     Arch,   med, 

navale,  1879,  xxxii,  p.  211. 


244  REFERENCES 

OSLER,   W.     On   Amoebic   Abscess    of   the    Liver.     Med.    News, 
1902,  15,  p.  673. 

PATTERSON,  H.  S.     Endemic  Amoebic  Dysentery  in  New  York, 

with  a  Review  of  its  Distribution  in  North  America.     Am. 

Jour.  Med.  Sci.,  1909,  cxxxviii,  p.  198. 
PFUHL.     Archives  de  Medicine  Navale,  1906,  p.  401. 
PLEHN,  A.     Die  Dysenteric  in  Kamerun.     Arch,  f .  Schiffs-  u. 

Tropen-Hyg.,  1898,  p.  125. 
PROWAZEK,  S.     Entamceba   buccalis.   n.   sp.   Arb.    a.    d.   kaiserl. 

Gesundheitsamte,   1904,  xxi,  p.   42. 

QUINCKE     and     Roos.     Ueber     amoeben-Enteritis.     Berl.     klin. 
Wochenschr.,  1893,  xxx,  p.  1089. 

ROGERS,    L.     Tropical    or   Amoebic    Abscess    of   the    Liver,    etc. 

Brit.   Med.   Jour.,   1902,   No.   2177,  p.   844. 
RUGE,    A.     Amoebenruhr.    Handbook    d.    Tropsmkrank.    Mense, 

1906,  iii,  p.   1. 

SCHARDINGER.     Protozoenkulturen.     Centralbl.     f.     Bakt.,     etc., 

1897,  1  Abt.  xxii,  p.  3. 
SCHAUDINN,   F.     Ueber  den  Zengungskreis   von   Paramoeba   eil- 

hardi;    neue  Genus,  neue  Species.     Sitzungst.  d.  h.  preuss. 

akad.  d.  Wissensch.,  Berlin,  1896,  p.  31. 
Idem.     Untersuch.  ueber  d.  Fortplanzung.  d.  Rhizopoden.     Art. 

a.  d.  Kaiserl.  Gesundheitsamte,  1903,  xix,  3,  p.  547. 
SCHUBERG,     A.     Die     parasit.     Amosben     d.     menschl.     Darms. 

Centralbl.  f.  Bakt.,  1893,  xiii,  p.  598;    p.  654;    p.   701. 
STILES,  C.  W.     Report  on  Classification  of  Amoeba  coli.     Am. 

Public  Health  Assoc.   Rep.,   1905,  xxx,  p.   292. 


REFERENCES  245 

STRONG,  R.,  and  MUSGRAVE,  W.  E.  Etiology  of  the  Dysenteries 
of  Manila.  Ann.  Rep.  Surg.  General,  U.  S.  Army,  1900, 
p.  251. 

STRONG,  R.  P.  Amoebic  Dysentery.  "  Modern  Medicine,"  Osier, 
Vol.  I.  Phila.,  1907. 

TSUJITANI.  Ueber  die  Reinkultur  des  Amoeben.  Centralbl.  f. 
Bakt.,  etc.,  1898,  1  Abt.  xxiv,  p.  666. 

VEDDER,  E.  B.  An  Examination  of  the  Stools  of  One  Hundred 
Healthy  Individuals,  with  Special  Reference  to  the  Pres- 
ence of  Entamoeba  coli.  Jour.  Am.  Med.  Assoc.,  1906, 
xxvi,  p.  870. 

Idem.  Is  the  Distinction  between  E.  coli  and  E.  dysenteriae 
valid?  Jour.  Trop.  Med.,  1907,  x,  p.  190. 

Idem.  Efficacy  of  the  Ipecac  Treatment  of  Dysentery.  Bull. 
Manila  Med.  Soc.,  1911,  HI,  3,  p.  48. 

VIERECK,  H.  Studien  ueber  die  in  den  Tropen  erworbene 
Dysenteric.  Archiv  f.  Schiffs-  u.  Tropen-Hyg.,  1907,  Bd. 
xi,  Beihest  I,  pp.  1-41. 

WALKER,  E.  L.    The  Parasitic  Amoebae  of  the  Intestinal  Tract  of 

Man  and  Other  Animals.     Jour.  Med.  Research,  1908,  xvii, 

p.  379. 
WEN  YON,  C.  M.     Reports  Wellcome  Research  Laboratories,  1908,, 

iii,  p.  122. 
WERNER,  H.      Studies   Regarding   Pathogenic  Amoebae.      Indian 

Med.  Gazette,  1909,  xliv,  p.  241. 
Idem.     Studien    iiber    pathogene    amoben.     Arch.    f.    Schiff-    u. 

Tropen-Hyg.,  1909,  Bd.  xii,  Beihest,  11,  p.  18. 
WILLIAMS,  A.  W.,  and  GURLEY,  C.  R.     Studies  Research  Lab., 

Dept.  of  Health,  N.  Y.  City,  1908-09,  vol.  iv,  p.  237. 


246  REFERENCES 

WOOLEY,  P.  G.,  and  MUSGRAVE,  W.  E.     The  Pathology  of  In- 
testinal Amoebiasis.    Jour.  Am.  Med.  Assoc.,  1905,  p.  1371. 

ZANCAROL.     Pathogenic  des  absces  du  foie.     Rev.  Centralbl.  f. 

Bakt.,  etc.,  1893,  1  Abt.  xiv,  p.  638. 
ZAUBITZER,  H.     Studien  ueber  eine  dom  Strohinfus  entaommene 

amoebe.     Arch.  f.  Hyg.,  1901,  xlii,  p.  311. 


INDEX  OF  AUTHORS 


A. 

Anderson,  114. 
Artault,  37,  235. 
Ashburn,  77,  80,  82,  112. 

B. 

Babes,  83. 

Baelz,  37,  233. 

Barbagallo,  7,  29,  32,  34,  36,  62,  64, 

83,  100,  102. 
Bensen,  180. 
Berndt,  83. 
Billet,  237. 
Blanchard,  3. 
Boggs,  114. 
Braun,  59. 
Brown,  W.  C.,  237. 
Butschli,  15. 
Buxton,  160. 

C. 

Cahen,  6. 

Calandruccio,  3. 

Calkins,  9. 

Cambay,  154. 

Casagrandi,  7,  29,  32,  34,  36,  62,  64, 

83,  100,  102. 
Castellani,  37,  73,  214. 
Celli,  7,  32,  83,  100,  108. 
Clegg,  7,  35,  62,  63,  64,  65,  66,  71, 

89,  163,  190,  203. 

Councilman,  5,  6,  31,  35,  151,  159. 
Cunningham,  2,  3,  61,  74,  83. 

D. 

Delafield,  47. 
Dessy,  114. 
Dock,  5,  75,  114. 
Doflein,  9,  36,  63,  216. 
Dopter,  239. 
Dutroleau,  154. 
Duncan,  114. 

E. 

Ehrenberg,  34. 

Elmassian,  10,  36,  73,  200,  206,  207. 

Ellis,  114. 


F. 

Fantham,  9. 

Fearnside,  114. 

Fiocca,  7,  32,  83,  100,  108. 

Flexner,  S.,  234. 

Frosch,  62. 

Futcher,  159. 

G. 

Gassard,  75. 

Gasser,  240. 

Gauducheau,  36,  73,  213. 

Gensen,  64. 

Giemsa,  46,  49. 

Grassi,  3,  37,  61,  74,  83,  232. 

Gros,  37,  83,  232. 

Gurley  (Williams  and  Gurley),  245. 

H. 

Harris,  7,  26,  176. 

Hartmann,  vi,  vii,  9,  10,  71,  72,  180, 

182,  188,  190,  191. 
Heidenhain,  48,  57. 
Hlava,  5,  114. 
Haspel,  154. 
Howard,  154. 

I. 

Ijima,  37,  83,  234. 

J. 

Jaeger,  241. 
Jennings,  21. 

Jiirgens,  7,  9,  33,  34,  109,  114,  149, 
233. 

K. 

Kartulis,  4,  5,  9,  61,  64,  83,  114,  151, 

233,  234. 
Koch,  3. 
Koidzumi,  10,  36,  73,  207,  208,  210, 

212. 

Kovacs,  6,  108. 
Kruse,  6,  31,  83,  108,  153. 


247 


248 


INDEX  TO  AUTHORS 


Lafleur,  5,  6,  31,  35,  114,  151,  159. 

Lambl,  1,  2,  11. 

Lesage,  10,  36,  73,  212,  213. 

Lewis,  2. 

Lcesch,  2,  36,  73. 

Liihe,  9,  59,  63. 

Lutz,  6. 

Long,  115. 

M. 

Mallory,  54,  57. 

Marchoux,  114. 

Marshall,  243. 

Marrotta,  114. 

Massiutin,  5,  83. 

Miller,  64. 

Minchin,  9. 

Miura,  243. 

Mouton,  62. 

Musgrave,  7,  26,  33,  35,  62,  63,  64, 

65,  66,  67,  68,  69,  71,  75,  80,  89, 

163,  190,  203. 
Musser,  5,  114. 


N. 


Noc,  179. 
Nakagawa,  114. 
Normand,  3,  83. 


Ogata,  64. 
Osier,  5,  114. 


O. 


P. 


Pasquale,  6,  31,  83,  108,  159. 
Patterson,  114. 
Perroncito,  383. 
Pfeiffer,  5. 
Pfuhl,  114. 
Plehn,  A.,  114. 
Posner,  233. 
Powell,  114. 
Prout,  114. 

Prowazek,  10,  36, 180,  182, 190, 191, 
230. 

Q. 

Quincke,  6,  30,  83. 


R. 

Rogers,  L.,  114. 
Romanowsky,  16,  43,  96. 
Roos,  6,  30,  83. 
Rouis,  153,  154. 
Ruge,  114. 

S. 

Schardinger,  62. 

Schaudinn,  vi,  vii,  2,  8/9,  11,  28,  33, 
34,  35,  36,  43,  63,  73,  75,  82,  90, 
100,  101,  105,  109,  112,  114,  122, 
133,  135,  137,  141,  162,  163,  165, 
215,  216,  222. 

Shuberg,  74,  83. 

Siler,  181. 

Simon,  9. 

Smith,  C.,  151. 

Sonsino,  3. 

Stengel,  5. 

Stiles,  9,  34,  35. 

Strong,  7,  33,  75,  108,  114. 


Tsujitani,  62. 
Tuttle,  114. 


T. 


V. 


73,  114, 


Vedder,  27,  77,k82,  114. 
Viereck,  vii,  9,  10,  36,  ( 

180,  182,  190,  191. 
Vivaldi,  64. 

W. 

Walker,  63,  67,  68,  69,  70,  71,  190. 

Waring,  154. 

Wellman,  114. 

Wenyon,  9. 

Werner,  vii,  9,  134,  .138,  141,l{178, 

190,  191,  192. 
Whitmore,  71,  82. 

Williams  (Williams  and  Gurley ),  245 . 
Woolly,  114. 
Wright,  43,  46,  51,  95,  96. 


Z. 


Zancarol,  151. 
Zaubitzer,  62,  64. 


GENERAL  INDEX 


A. 

Abscess,  of  liver,  3,  151. 

experimental  production  of, 
172. 

location  of,  152. 

number  of,  152. 

pathology  of,  155. 

percentage  of,  151. 

rupture  of,  154. 
Acetic  acid  solution,  45. 
Alcohol,  in  fixation,  44. 
Amoebae, 

biology  of,  19. 

classification  of,  28. 

cultivation  of,  58. 

history  of,  1,  12,  34,  73,  100, 
108,  114,  179,  200,  207,  212, 
215,  230. 

morphology  of,  12. 

pathogenicity  of,  6,  108,  162, 
191,  211,  213,  227,  232. 

resistance  of,  26. 

reproduction  of ,  13, 23, 100, 133, 
187,  210,  213,  222,  226,  231. 
Amoeba  coli,  30,  31,  33. 

coli  mitis,  30. 

diaphana,  32. 

dysenteriae,  6,  31,  33,  35. 

intestini  vulgaris,  30. 

limax,  17. 

lobosa,  var.  guttata,  32. 
oblonga,  32. 

proteus,  17,  18. 

reticularis,  32. 

spinosa,  32. 

vermicularis,  32. 
Argyrol,  27. 

B. 

Bacillary  dysentery,  144. 
Bacteria  and  dysentery,  144,  146. 

liver  abscess,  159. 
Bacillus  coli,  65,  67. 


Bacillus  fluorescens,  65. 
rubra,  65. 
typhosus,  65. 

C. 

Carbol  fuchsin,  47,  48. 

Cats,  production   of    dysentery  in, 

108,  109,  163,  166. 
Centriole,  16,  231. 
Centrosome,  16,  222. 
Cholera  and  amoebae,  83. 
Chromatin,  16. 

distribution  of,  16. 
in  E.  buccalis,  231. 
coli,  94. 
histolytica,  124. 
minuta,  204. 
nipponica,  207. 
tetragena,  184,  187. 
tropicalis,  212. 
Paramoeba  hominis,  218. 
Chromidia,  15,  16,  139,  187. 
Classification  of  amoebae,  28. 
Copper  sulphate,  27. 
Cultivation  of  amoebae,  58,  61. 

of  different  species,  59,  63,  107, 

179,  190,  213. 
technique  of,  64. 
Conjugation,  25,  141,  189. 
Cysts  of  E.  coli,  100. 

histolytica,  134. 
minuta,  205. 
nipponica,  211. 
tetragena,  188. 

Cysts  of  Paramoeba  hominis,  219 
Cytoplasm,  14. 

of  Entamoeba  buccalis,  231. 
coli,  90. 

histolytica,  121. 
minuta,  201. 
nipponica,  208. 
tetragena,  183. 
tropicalis,  212. 
Paramoeba  hominis,  218. 


249 


250 


GENERAL  INDEX 


D. 

Diagnosis,  methods  of,  39,  47,  193. 
of  Entamoeba  coli,  111,  193. 
histolytica,  118,  193. 
tetragena,  193. 
differential,  193. 

Diarrhoea,  amoebae  in,  3,  83,  110. 
Disease,  relation  of  amoebae  to,  107, 
142,   162,   191,  206,j211,   213, 
227,  232. 

Distribution,  geographical,  76. 
of  E.  buccalis,  230. 
coii,  76. 

histolytica,  114. 
minuta,  200. 
nipponica,  207. 
tetragena,  182. 
tropicalis,  212. 
Paramoeba  hominis,  217. 
Dysentery,  amoebic,  1,  110,  192. 
bacillary,  144. 
history  of,  1. 
E.  coli  and,  110,  112, 

histolytica   and,    110,    114, 

142,  162. 

tetragena  and,  191. 
other    amoebae    and,    206,  211, 

213. 

Experimental  production  of,  3,  5,  7, 
8,  108,  162,  166,  170. 

E. 

Ectoplasm,  14. 

structure  of,  14,  97. 
Endoplasm,  14. 

structure  of,  14,  97. 
Entamoeba  buccalis,  230. 

distribution  of,  230. 

history  of,  230. 

morphology  of,  231. 

motility  of,  231. 

relation  to  disease  of,  232. 

reproduction  of,  231. 
Entamoeba  coli,  73. 

color  of,  88. 

conjugation  in,  25. 

cultivation  of,  60,  107. 

cytoplasm  of,  90. 

diagnosis  of,  111,  193. 

ectoplasm  of,  14,  90. 

endoplasm  of,  91. 

erythrocyles  in,  93. 

history  of,  8,  32,  34,  73, 100, 108. 


Entamoeba  coli,  morphology  of,  86. 

motility  of,  99. 

nucleus  of,  93. 

occurrence  in  health,  74. 

pseudopodia  of,  22,  99. 

relation  to  disease,  108. 

reproduction  of,  100. 

shape  of,  88. 

size  of,  87. 

spores  of,  18. 

staining  of,  95. 

vacuoles  in,  17,  92. 
Entamoeba  dentalis,  232. 
Entamoeba  histolytica,  114. 

color  of,  120. 

conjugation  in,  25,  141. 

cultivation  of,  60,  61,  63,  179. 

cytoplasm  of,  121. 

diagnosis  of,  118,  193. 

ectoplasm  of,  14,  121. 

endoplasm  of,  122. 

erythrocytes  in,  126. 

history  of,  6,  9,  34,  114. 

morphology  of,  115. 

motility  of,  128. 

nucleus  of,  123. 

pseudopodia  of,  22,  128. 

relation  to  disease,  162. 

reproduction  of,  13,  23, 101, 133. 

shape  of,  119. 

size  of,  116. 

spores  of,  19,  133. 

staining  of,  132. 

vacuoles  in,  17,  125. 
Entamoeba  kartulisi,  234. 
Entamoeba  minuta,  200. 

cytoplasm  of,  201. 

history  of,  200. 

morphology  of,  200. 

motility  of,  205. 

nucleus  of,  204. 

relation  to  disease,  206. 

reproduction  of,  205. 

shape  of,  201. 

size  of,  200,  201. 

vacuoles  in,  204. 
Entamoeba  miurai,  234. 
Entamoeba  nipponica,  207. 

cytoplasm  of,  208. 

history  of,  207. 

morphology  of,  207. 

motility  of,  210. 

nucleus  of,  209. 

relation  to  disease,  211. 


GENERAL  INDEX 


251 


Entamoeba  nipponica,  reproduction 
of,  210. 

shape  of,  208. 

size  of,  208. 

vacuoles,  209. 

Entamoeba  phagocytoides,  213. 
Entamoeba  pulmonalis,  235. 
Entamoeba  tetragena,  179. 

conjugation  in,  25,  189. 

cultivation  of,  60,  190. 

cytoplasm  of,  183. 

history  of,  6,  10,  179. 

morphology  of,  182. 

diagnosis  of,  193. 

motility  of,  186. 

nucleus  of,  184. 

relation  to  disease,  191. 

reproduction  of,  187. 

shape  of,  183. 

size  of,  183. 

staining  of,  186. 

vacuoles  in,  185. 
Entamoeba  tropicalis,  212. 

cultivation  of,  213. 

history  of,  212. 

morphology  of,  212. 

relation  to  disease,  213. 
Entamoeba  urogenitalis,  233. 

undulans,  214. 
Erythrocytes,  93. 

in  E.  coli,  93. 

histolytica,  120,  126. 
tetragena,  185. 
Eosin  and  methylene  blue,  55. 

F. 

Faeces,  cultures  from,  66. 

examination  of,  39,  47. 
Flagella,  23,  224. 
Fixation,  technique  of,  44,  47. 


Gemmation  in  E.  histolytica,  19,133. 
Gentian  violet,  47,  48. 
Giemsa  stain,  49. 

H. 

Haematoxylin,  47. 
Hepatitis,  and  amoebae,  83. 
Hydrogen  dioxide,  27. 
Heidenhain's  iron  hsematoxylin,  48, 
57. 


I. 

Idiochromidia,  16. 

Incubation,  period  of,  in  experi- 
mental dysentery,  164,  168,  171, 
192. 

Intestine,  amoebae  of,  73. 
lesions  of,  4,  145. 
ulcers  of,  3,  146. 

Ipecac,  action  of,  27. 

Iron  hsematoxylin  staining  method, 
48,  57. 

K. 

Karyosome,  15,  16. 

of  E.  coli,  94,  95,  98. 

histolytica,    116,    124, 

133. 

minuta,  204, 
nipponica,  209. 
tetragena,  185,  187. 
tropicalis,  212. 
Paramoeba  hominis,  220. 


L. 

Liver,  abscess  of,  3,  151. 

lesions  of,  in  dysentery,  3,  151 
pathology  of,  155. 

M. 

Mallory's  staining  method,  54. 
Mercuric  chloride,  44. 
Methylene  blue,  47,  48. 
Microscope,  incubator,  40. 
Microsomes,  15. 
Mitosis,  in  E.  coli,  101,  104,  106. 

histolytica,  134. 

tetragena,  187. 
Morphology  of  E.  buccalis,  231. 

coli,  86. 

histolytica,  115. 

minuta,  200. 

nipponica,  207. 

tetragena,  182. 

tropicalis,  212. 
Paramoeba  hominis,  218. 
Motility  of  E.  buccalis,  231. 

coli,  99. 

histolytica,  128. 

minuta,  205. 

nipponica,  210. 


252 


GENERAL  INDEX 


Motility  of  E.  tetragena,  186. 

tropicalis,  212. 
Paramoeba    hominis,   220, 

224. 
Mouth,  amoebae  of,  230. 

N. 

Nucleus,  structure  of,  15. 

achromatic  substance  of,  16. 
centrosome  of,  16. 
centriole  of,  16. 
chromidia  of,  15. 
chromatin  of,  16. 
karyosome  of,  15,  16,  94,  95,  98, 
116,  124,  133,  185,  187,  204, 
209,  212,  220. 

reproductive  changes  in,  24,  25, 
93,  101,  102,  104,  133,  184, 
204,  221. 

of  E.  buccalis,  231. 
coli,  93. 

histolytica,  123. 
minuta,  204. 
nipponica,  209. 
tetragena,  184. 
tropicalis,  212. 
Paramoeba   hominis,    220, 

223,  224. 

staining  of,  95,  132,  186. 
Neutral  red,  42. 
Nomenclature,  28. 
Nutrition  of  amoebae,  19. 

O. 

Osmic  acid,  fixation  by,  44,  46. 

P. 

Paramoeba  hominis,  amoebic  stage 
of,  220. 

diagnosis  of.  228. 

distribution  of,  217. 

encysted  stage  of,  223. 

flagellate  stage  of,  224. 

history  of,  215. 

life  cycle  of,  216,  218. 

morphology  of,  218. 

relation  to  disease  of,  227. 

reproduction  of,  227. 
Paramoeba  eilhardi,  29. 
Pathology  of  dysentery,  144. 

of  liver  abscess,  151,  155. 

microscopic,  149. 
Permanganate  of  potassium,  27. 


Picric  acid,  45. 
Pseudopodia  of  E.  coli,  99. 
of  E.  histolytica,  128. 
tetragena,  186. 

Q. 

Quinine  sulphate  and  amoebae,  27. 

R. 

Reproduction  by  cyst  formation,  25, 

100,  134,  187,  205,  210. 
by  gemmation,  24,  134. 

schizogony,  24, 100,  205,  209. 
simple    division,     24,     100, 
101,  134,  187,  205,  209. 
of  E.  buccalis,  231. 
coli,  100. 
histolytica,  13,  23,  101, 

133. 

minuta,  205. 
nipponica,  210. 
tetragena,  187. 
tropicalis,  212. 
Paramoeba  hominis,  222. 
Rontgen  rays,,  27. 

S. 

Sections,  staining  of,  53. 
Schizogony,  description  of,  24. 
in  E.  buccalis,  231. 
coli,  100. 
histolytica,  133. 
minuta,  205. 
nipponica,  210. 
Silver  nitrate,  27. 
Sporogony  in  E.  histolytica,  134. 
Spores,  relation  to  disease  of,  163. 

of  E.  histolytica,  134. 
Stage,  warm,  40. 
Staining  methods,  47. 

carbol  fuchsin,  47,  48. 
eosin  and  methylene  blue,  55. 
Giemsa  stain,  49. 
hsematoxylin,  Delafield's,  47. 
haematoxylin-eosin  stain,  56. 
iron    haematoxylin,  method    of 

Heidenhain,  48,  57. 
safranin  stain,  54. 
Wright  stain,  51. 
Stimulation,  response  of  amoebae  to,. 

20. 

Sublimate  acetic  acid  mixture,  45. 
Sulphate  of  copper,  27. 


GENERAL  INDEX 


253 


T. 

Technique,  38. 

of  examination  of  living  amoebae, 

39. 

fixation,  44,  47. 
staining,  43,  47. 
staining  sections,  53. 
Typhoid,  amoebae  in,  83. 

U. 

Ulcers  of  Intestine,  145. 


V. 

Vacuoles,  17. 

contractile,  17. 

cultural  amoebae  and,  17. 

digestive,  18. 

formation  of,  18. 

size  of,  17. 

in  E.  coli,  92. 

histolytica,  125. 

tetragena,  185. 

W. 

Water,  cultures  of  amoebae  from,  66. 


THIS  BOOK  IS  DUB  ON  THE  LAST  DATE 
STAMPED  BELOW 

AN  INITIAL  FINE  OF  25  CENTS 

WILL  BE  ASSESSED  FOR  FAILURE  TO  RETURN 
THIS  BOOK  ON  THE  DATE  DUE.  THE  PENALTY 
WILL  INCREASE  TO  SO  CENTS  ON  THE  FOURTH 
DAY  AND  TO  $1.OO  ON  THE  SEVENTH  DAY 
OVERDUE. 


FEB  2  3  REC'Q 


L1'  :•- 
luu" r\  i 

JUN 

.lM«i-        MflY29REC'° 

jpCT  1 7  1963 
NOV  1 7 1964 

UCD  LIBRARY 

)UE  JUN  1 0  196 

MAY  3 1  REC'D 

LJRRARY,    BRANCH    OF    THE    COLLEGE    OF   AGRICULTURE 

5m-9,'35(s) 


o 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


